JP6677659B2 - Improved amino acid sequence directed to human respiratory syncytial virus (hRSV) for prevention and / or treatment of respiratory tract infection and polypeptide containing the same - Google Patents

Description

  The present invention provides amino acid sequences that are directed and / or can specifically bind to protein F of hRSV (as defined herein), as well as one or more such amino acids. A compound or construct comprising, or consisting essentially of, a sequence, in particular proteins and polypeptides (each referred to herein as “amino acid sequence (s) of the invention”, “compound (s) of the invention” , "" The construct (s) of the invention "and" the polypeptide (s) of the invention ").

  The invention relates to nucleic acids encoding such amino acid sequences and polypeptides (also referred to herein as "nucleic acid (s) of the invention" or "nucleotide sequence (s) of the invention"). In a method of preparing such amino acid sequences and polypeptides, a host cell expressing or capable of expressing such amino acid sequences or polypeptides may be provided with such amino acid sequences, polypeptides, compounds or constructs, Compositions comprising nucleic acids and / or host cells, especially pharmaceutical compositions, and such amino acids for prophylactic and / or therapeutic purposes, such as the prophylactic and / or therapeutic purposes specifically referred to herein. It also relates to the use of sequences, polypeptides, compounds or constructs, nucleic acids, host cells and / or compositions.

  Other aspects, embodiments, advantages and uses of the present invention will become apparent from the further description herein.

  Human respiratory syncytial virus (hRSV) is a member of the Paramyxviridae family and is an enveloped virus with two major surface glycoproteins that make up the spike of virions. One of these glycoproteins (protein G) is an adhesion protein that mediates the binding of the virus to the cell surface. Other glycoproteins (Protein F or fusion proteins) mediate fusion of the viral and cellular membranes, allowing the viral nucleocapsid to enter the cytoplasm. Inhibition of processes mediated by either G- or F-glycoprotein blocks the early stages of the infection cycle and neutralizes the infectivity of the virus. Therefore, an antibody that has a tropism for either the G glycoprotein or the F glycoprotein and inhibits the activity of each can neutralize the infectivity of the virus and prevent hRSV infection. The F protein is highly conserved and forms trimeric spikes that undergo structural changes upon activation.

  hRSV is the leading cause of severe lower respiratory tract infections (bronchiolitis and pneumonia) in infants and infants and is responsible for the winter epidemic every year. The virus also causes a significant disease burden in the elderly, and there is also a risk of severe hRSV disease in adults with cardiopulmonary disorders and / or immunosuppression. Reinfection is not prevented by the immune response.

  No vaccine is available to prevent hRSV infection. The only formulation on the market is a humanized monoclonal antibody directed against one of the viral glycoproteins (Protein F) (Synazis®), which has a very high risk of developing severe hRSV infection. Used prophylactically in high children. Synagis® is at least partially restricted in use due to the high cost of its products. Clearly, there is a need for improved and / or cheaper prophylactic and / or therapeutic agents for the prevention and / or treatment of infections with hRSV.

  The present invention is directed to protein F of hRSV and has improved prophylactic, therapeutic and / or pharmacological properties compared to prior art amino acid sequences and antibodies, as well as other beneficial properties. Amino acid sequence (also referred to as “amino acid sequence (s) of the present invention”) and polypeptides (“the present invention”) having various properties (eg, improved ease of preparation and / or reduced product cost). Polypeptide (s) ", as well as therapeutic compounds and compositions. These improved beneficial properties will become clear from the further description herein. Without limitation, the amino acid sequences, polypeptides, and therapeutic compounds and compositions provided by the present invention provide improved stability, reduced immunogenicity, and improved binding of hRSV to protein F. Improve the affinity and / or avidity of hRSV for protein F, improve the efficacy and / or efficacy of neutralizing hRSV (as defined herein), increase the selectivity of hRSV for protein F. And / or they can partially or preferably completely block the interaction of protein F of hRSV with the target host cell and / or its membrane. They modulate, inhibit and / or block hRSV infectivity, modulate, inhibit and / or block fusion of hRSV to (the cell membrane of) target host cells, and / or (see herein). By regulating, inhibiting and / or preventing hRSV entry in the targeted host cell (as defined herein), hRSV can be neutralized. They may be cross-reactive with and / or neutralize different strains of hRSV and / or different hRSV escape mutants.

  In a first aspect, the invention provides a number of amino acid residue stretches (as defined herein) that are particularly suitable for binding to a particular epitope on protein F of hRSV. These stretches of amino acid residues may be present in and / or incorporated into the amino acid sequence of the invention, particularly to form (part of) the antigen binding site of the amino acid sequence of the invention. The resulting amino acid sequence is at antigenic site II on protein F of hRSV (ie, amino acid residues 250-275 of protein F of hRSV), forms part of antigenic site II on protein F of hRSV, or On protein F of hRSV that overlaps (ie, in primary or tertiary structure) with or close to antigen site II on protein F of hRSV (ie, in primary or tertiary structure) It is possible to bind to a specific epitope of

Thus, in one aspect, the invention provides:
a) SEQ ID NO: 102,
b) a stretch of amino acid residues having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 102,
i) the amino acid residue stretch has aspartic acid (Asp, D) at position 6 (position 54 determined by Kabat numbering),
ii) the amino acid sequence comprising the amino acid residue stretch has the same, substantially the same or higher affinity as compared to the amino acid sequence comprising the amino acid residue stretch having no three, two or one amino acid difference ( The affinity is as measured by surface plasmon resonance) and binds to protein F of hRSV and / or the amino acid sequence comprising the amino acid residue stretch has three, two or one amino acid differences. An amino acid selected from an amino acid residue stretch that has the same, approximately the same, or a higher potency (as defined herein) as compared to an amino acid sequence comprising the amino acid residue stretch without An amino acid sequence comprising at least one stretch of residues is provided.

In a preferred embodiment, the present invention comprises two or more stretches of amino acid residues, one stretch comprising:
a) SEQ ID NO: 102,
b) a stretch of amino acid residues having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 102,
i) the amino acid residue stretch has aspartic acid (Asp, D) at position 6 (position 54 determined by Kabat numbering),
ii) the amino acid sequence comprising the amino acid residue stretch has the same, substantially the same or higher affinity as compared to the amino acid sequence comprising the amino acid residue stretch having no three, two or one amino acid difference ( The affinity is as measured by surface plasmon resonance) and binds to protein F of hRSV and / or the amino acid sequence comprising said stretch of amino acid residues differs by three, two or one amino acid Is selected from amino acid residue stretches that have the same, approximately the same, or higher potency (as defined herein) as compared to the amino acid sequence comprising the amino acid residue stretch without
At least one stretch,
c) SEQ ID NO: 98;
d) An amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 98, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (as said affinity is measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference the amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; An amino acid residue stretch, which shall have about the same or higher potency (as defined herein);
e) an amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 121, said amino acid sequence comprising said amino acid residue stretch Has the same, nearly the same, or a higher affinity compared to an amino acid sequence comprising the amino acid residue stretch without three, two or one amino acid difference (the affinity is measured by surface plasmon resonance An amino acid sequence that binds to protein F of hRSV and / or contains an amino acid residue stretch that does not have three, two or one amino acid difference Amino acids that have the same, about the same, or higher potency (as defined herein) as compared to the sequence The amino acid residue stretch selected from the amino acid residue stretch and corresponding to one of a) and b) is such that it is always present in the amino acid sequence of the invention, and the second amino acid residue stretch is An amino acid sequence is provided which is as selected from one of c), d), e) and f).

Even more preferably, the amino acid sequence of the invention comprises three or more amino acid residue stretches, wherein the first amino acid residue stretch comprises
a) SEQ ID NO: 98,
b) An amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 98, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (such as measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference an amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Consisting of stretches of amino acid residues, which should have about the same or higher potency (as defined herein) It is selected from,
The second amino acid residue stretch is
c) SEQ ID NO: 102,
d) a stretch of amino acid residues having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 102,
i) the amino acid residue stretch has aspartic acid (Asp, D) at position 6 (position 54 determined by Kabat numbering),
ii) the amino acid sequence comprising the amino acid residue stretch has the same, substantially the same or higher affinity as compared to the amino acid sequence comprising the amino acid residue stretch having no three, two or one amino acid difference ( The affinity is as measured by surface plasmon resonance) and binds to protein F of hRSV and / or the amino acid sequence comprising the amino acid residue stretch has three, two or one amino acid differences. Selected from the group consisting of amino acid residue stretches that have the same, approximately the same, or higher potency (as defined herein) as compared to an amino acid sequence comprising the above amino acid residue stretches without And
The third amino acid residue stretch is
e) an amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 121, said amino acid sequence comprising said amino acid residue stretch Has the same, nearly the same, or a higher affinity compared to an amino acid sequence comprising the amino acid residue stretch without three, two or one amino acid difference (the affinity is measured by surface plasmon resonance An amino acid sequence that binds to protein F of hRSV and / or contains an amino acid residue stretch that does not have three, two or one amino acid difference Amino acids that have the same, about the same, or higher potency (as defined herein) as compared to the sequence Noic acid residue stretch.

One of these specific amino acid residues stretches or comprising the amino acid sequence, for example (as further described herein (actual or apparent) K _D values (actual or apparent) K _A values, k _on rate and / or _{k off} rate, or alternatively is preferably measured as _{IC 50} values, and / or represented by) improved binding characteristics, improved affinity for a protein F of hRSV and / or avidity And / or improved properties such as improved efficacy and / or efficacy of neutralizing hRSV.

The amino acid sequences of the present invention are particularly domain antibodies (or amino acid sequences suitable for use as domain antibodies), single domain antibodies (or amino acid sequences suitable for use as single domain antibodies), "dAbs" (or dAbs). Amino acid sequences suitable for use as), or Nanobodies® (including but not limited to _VHH sequences as defined herein), other single variable domains, or any of them It can be any one suitable fragment.

In this regard, the amino acid sequences of the invention may consist essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), where CDR2 But,
a) a stretch of amino acid residues having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 102,
i) the amino acid residue stretch has aspartic acid (Asp, D) at position 6 (position 54 determined by Kabat numbering),
ii) the amino acid sequence comprising the amino acid residue stretch has the same, substantially the same or higher affinity as compared to the amino acid sequence comprising the amino acid residue stretch having no three, two or one amino acid difference ( The affinity is as measured by surface plasmon resonance) and binds to protein F of hRSV and / or the amino acid sequence comprising the amino acid residue stretch has three, two or one amino acid differences. Selected from amino acid residue stretches that have the same, approximately the same, or higher potency (as defined herein) as compared to an amino acid sequence that includes the above amino acid residue stretches having no.

  These preferred complementarity determining regions (CDR2 sequences) are also referred to as "CDR2 (s) of the invention".

Preferably, the amino acid sequence of the invention may consist essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), where CDR2 But,
a) a stretch of amino acid residues having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 102, or b) SEQ ID NO: 102;
i) the amino acid residue stretch has aspartic acid (Asp, D) at position 6 (position 54 determined by Kabat numbering),
ii) the amino acid sequence comprising the amino acid residue stretch has the same, substantially the same or higher affinity as compared to the amino acid sequence comprising the amino acid residue stretch having no three, two or one amino acid difference ( The affinity is as measured by surface plasmon resonance) and binds to protein F of hRSV and / or the amino acid sequence comprising the amino acid residue stretch has three, two or one amino acid differences. Selected from the group consisting of amino acid residue stretches that have the same, approximately the same, or higher potency (as defined herein) as compared to an amino acid sequence comprising the above amino acid residue stretches without And
At least one of CDR1 or CDR3 is
c) SEQ ID NO: 98;
d) An amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 98, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (as said affinity is measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference the amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Consisting of stretches of amino acid residues, which should have about the same or higher potency (as defined herein) And / or e) SEQ ID NO: 121, or f) a stretch of amino acid residues having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 121, The amino acid sequence comprising the amino acid residue stretch has the same, substantially the same, or a higher affinity (the affinity) as compared to the amino acid sequence comprising the amino acid residue stretch having no three, two or one amino acid difference. Sex is as measured by surface plasmon resonance) and binds to protein F of hRSV and / or the amino acid sequence comprising said amino acid residue stretch has three, two or one amino acid differences. The same, nearly the same, or higher potency (as defined herein) as compared to an amino acid sequence comprising CDR3 selected from the group consisting of amino acid residue stretches
Is selected from

Even more preferably, the amino acid sequence of the present invention may consist essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively). In CDR1
a) SEQ ID NO: 98,
b) An amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 98, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (such as measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference an amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Consisting of stretches of amino acid residues, which should have about the same or higher potency (as defined herein) It is selected from,
CDR2 is
c) a stretch of amino acid residues having no more than 3, preferably no more than 2, and more preferably only 1 amino acid difference from SEQ ID NO: 102, or d)
i) the amino acid residue stretch has aspartic acid (Asp, D) at position 6 (position 54 determined by Kabat numbering),
ii) the amino acid sequence comprising the amino acid residue stretch has the same, substantially the same or higher affinity as compared to the amino acid sequence comprising the amino acid residue stretch having no three, two or one amino acid difference ( The affinity is as measured by surface plasmon resonance) and binds to protein F of hRSV and / or the amino acid sequence comprising the amino acid residue stretch has three, two or one amino acid differences. Selected from the group consisting of amino acid residue stretches that have the same, approximately the same, or higher potency (as defined herein) as compared to an amino acid sequence comprising the above amino acid residue stretches without And
CDR3 is
e) SEQ ID NO: 121,
f) an amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 121, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (such as measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference an amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Stretches of amino acid residues which should be of about the same or higher potency (as defined herein). It is selected from the group.

  In certain aspects, the amino acid sequence or Nanobody® of the invention comprises at least SEQ ID NO: 102. Preferably, the amino acid sequence or Nanobody® of the present invention comprises SEQ ID NO: 98, SEQ ID NO: 102 and SEQ ID NO: 121.

The present invention also provides a number of humanized amino acid sequences that are particularly suitable for binding to protein F of hRSV. The amino acid sequences of the present invention show reduced immunogenicity upon administration to a human subject. Furthermore, the amino acid sequences of the present invention, as compared to the corresponding wild type amino acid sequence, for example as described in (further herein with respect to protein F of hRSV (actual or apparent) K _D value, (in fact or apparent) _{K a value k on} rate and / or _{k off} rate, or alternatively is preferably measured as _{IC 50} values, and / or represented by) improved binding properties, affinity to protein F of hRSV Exhibits improved properties and / or improved avidity and / or other properties such as improved efficacy and / or potency to neutralize hRSV ("Amino acid sequences directed against envelope proteins of avirus and polypeptides comprising As described in International Application No. PCT / EP2009 / 056975, filed June 5, 2009, filed by Ablynx NV, entitled "the same for the treatment of viraldiseases". To).

Thus, in another aspect, the present invention provides:
a) SEQ ID NO: 60 to SEQ ID NO: 76
b) an amino acid sequence having only one amino acid difference from one of SEQ ID NO: 60 to SEQ ID NO: 76, preferably only two, more preferably only one;
i) the amino acid sequence has glutamine (Gln, Q) at position 105 (the position is determined by Kabat numbering);
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) (As defined herein) is provided.

  In a preferred aspect, the amino acid sequence of the invention comprises or consists essentially of SEQ ID NO: 60-SEQ ID NO: 76.

In another aspect, the invention provides:
a) SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76
b) only three, preferably only two and more preferably only one amino acid difference from one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75 and SEQ ID NO: 76 An amino acid sequence having
i) the amino acid sequence has glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and / or arginine (Arg, R) at position 83 (the above positions are determined by Kabat numbering; Desired),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) (As defined herein) is provided.

  A preferred amino acid sequence of the present invention comprises or consists essentially of one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76.

In yet another aspect, the invention provides a method comprising:
a) an amino acid sequence having only three, preferably only two, more preferably only one amino acid difference from one of SEQ ID NO: 65 and SEQ ID NO: 76;
i) the amino acid sequence is aspartic acid (Asp, D) at position 54, glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and / or arginine (Arg, R) at position 83; (The position is determined by Kabat numbering),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) (As defined herein) is provided.

  In a preferred embodiment, the amino acid sequence of the invention comprises or consists essentially of SEQ ID NO: 65. In another preferred embodiment, the amino acid sequence of the invention comprises or consists essentially of SEQ ID NO: 76.

In another aspect, the invention provides:
a) SEQ ID NO: 146 to SEQ ID NO: 153
b) an amino acid sequence having only one, preferably two, more preferably only one amino acid difference from one of SEQ ID NOs: 146 to 153,
i) the amino acid sequence has proline (Pro, P) at position 14, arginine (Arg, R) at position 19, leucine (Leu, L) at position 20, and leucine (Leu, L) at position 108 (The above position is determined by Kabat numbering),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) (As defined herein) is provided.

  In a preferred embodiment, the amino acid sequence of the invention comprises or consists essentially of SEQ ID NO: 146 to SEQ ID NO: 153.

In another aspect, the invention provides:
a) SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153,
i) the amino acid sequence is
SEQ ID NO: 146 (this amino acid sequence preferably has glutamine (Gln, Q) at position 105);
SEQ ID NO: 147 (this amino acid sequence preferably has arginine (Arg, R) at position 83 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 148 (this amino acid sequence preferably has glutamic acid (Glu, E) at position 85 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 149 (this amino acid sequence preferably has arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85 and glutamine (Gln, Q) at position 105), SEQ ID NO: 151 (this amino acid sequence Preferably has arginine (Arg, R) at position 83),
SEQ ID NO: 152 (this amino acid sequence preferably has glutamic acid (Glu, E) at position 85);
SEQ ID NO: 153 (this amino acid sequence preferably has arginine (Arg, R) at position 83 and glutamic acid (Glu, E) at position 85)
The amino acid sequence has a proline (Pro, P) at position 14 and an arginine (Arg, R) at position 19, as in the case of having only 3, preferably 2, and more preferably only 1 amino acid difference. Leucine (Leu, L) at position 20, Leucine (Leu, L) at position 108, arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85, and / or glutamine at position 105 (Gln, Q) (the position is determined by Kabat numbering),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) (As defined herein) is provided.

  Preferred amino acid sequences of the present invention comprise or consist essentially of one of SEQ ID NOs: 146-149 and SEQ ID NOs: 151-153.

The present invention provides a number of sequence-optimized amino acid sequences and / or Nanobodies® that show increased stability upon storage during stability testing and are particularly suitable for binding to protein F of hRSV. . The amino acid sequences of the present invention show reduced post-translational modifications of the pyroglutamate N-terminus and therefore increased product stability. Furthermore, the amino acid sequences of the present invention, as compared to the corresponding parent amino acid sequence, for example, reduced immunogenicity, as described (further herein with respect to protein F of hRSV (actual or apparent) K _D value (actual or apparent) _{K a value k on} rate and / or _{k off} rate, or alternatively is preferably measured as _{IC 50} values, and / or represented by) improved binding properties, of hRSV It exhibits improved affinity and / or improved binding activity for protein F and / or improved other properties such as improved efficacy and / or potency to neutralize hRSV ("Amino acid sequences directed against envelope proteins of" PCT / EP2009 / 056975 filed on June 5, 2009, filed by Ablynx NV entitled "avirus and polypeptides comprising the same for the treatment of viraldiseases" As the placing of).

Thus, in one aspect of the invention,
a) SEQ ID NO: 138 to SEQ ID NO: 141 and SEQ ID NO: 154 to SEQ ID NO: 157;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 138 to 141 and 154 to 157,
i) the amino acid sequence has aspartic acid (Asp, D) at position 1 (the position is determined by Kabat numbering);
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) An amino acid sequence and / or Nanobody® is provided, wherein the amino acid sequence is selected from the amino acid sequences (as defined herein).

  In a preferred embodiment, an amino acid sequence and / or Nanobody® of the invention comprises or consists essentially of one of SEQ ID NOs: 138-141 and SEQ ID NOs: 154-157.

  The amino acid sequences and Nanobodies® provided by the present invention may be in essentially isolated form (as defined herein) or may be a protein or polypeptide of the invention ("polypeptide of the invention"). (Also referred to as "peptide" or "protein of the invention") (which may comprise or consist essentially of one or more amino acid sequences or Nanobodies® of the invention, optionally one more Or to form part of a plurality of additional amino acid sequences or Nanobodies®, which may all comprise optionally linked via one or more suitable linkers.

  Thus, in another aspect, the present invention provides a protein or polypeptide comprising (or consisting essentially of) one or more amino acid sequences of the invention and / or Nanobodies® (or suitable fragments thereof). (Also referred to herein as "polypeptides of the invention").

  For example, but not limited to, one or more of the amino acid sequences and / or Nanobodies® of the present invention may be mono-, multi-, or multi-paratopic of the present invention as described herein. It can be used as a binding unit in such a protein or polypeptide to provide each polypeptide. The invention therefore also relates to polypeptides which are monovalent constructs comprising or consisting essentially of the amino acid sequences or Nanobodies® of the invention. Thus, the present invention also relates to polypeptides that are multivalent polypeptides, such as divalent or trivalent polypeptides. The invention also relates to polypeptides such as multiparatopic polypeptides, such as double or triple paratopic polypeptides.

  In a preferred embodiment, the invention comprises a polyvalent, preferably divalent or trivalent, comprising or consisting essentially of at least two amino acid sequences of the invention and / or Nanobodies® (as described above). Are provided.

In one aspect, the invention provides:
a) SEQ ID NO: 102,
b) a stretch of amino acid residues having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 102,
i) the amino acid residue stretch has aspartic acid (Asp, D) at position 6 (position 54 determined by Kabat numbering),
ii) the amino acid sequence comprising the amino acid residue stretch has the same, substantially the same or higher affinity as compared to the amino acid sequence comprising the amino acid residue stretch having no three, two or one amino acid difference ( The affinity is as measured by surface plasmon resonance) and binds to protein F of hRSV and / or the amino acid sequence comprising the amino acid residue stretch has three, two or one amino acid differences. An amino acid selected from an amino acid residue stretch that has the same, approximately the same, or a higher potency (as defined herein) as compared to an amino acid sequence comprising the amino acid residue stretch without At least two, preferably identical, amino acids selected from an amino acid sequence containing at least one residue stretch At least three (preferably identical) or comprising the amino acid sequence or Nanobody (TM), or which consists essentially of multivalent, preferably provides a divalent or trivalent polypeptide.

In another aspect, the invention provides that one stretch comprises:
a) SEQ ID NO: 102,
b) a stretch of amino acid residues having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 102,
i) the amino acid residue stretch has aspartic acid (Asp, D) at position 6 (position 54 determined by Kabat numbering),
ii) the amino acid sequence comprising the amino acid residue stretch has the same, substantially the same or higher affinity as compared to the amino acid sequence comprising the amino acid residue stretch having no three, two or one amino acid difference ( The affinity is as measured by surface plasmon resonance) and binds to protein F of hRSV and / or the amino acid sequence comprising said stretch of amino acid residues differs by three, two or one amino acid Is selected from amino acid residue stretches that have the same, approximately the same, or higher potency (as defined herein) as compared to the amino acid sequence comprising the amino acid residue stretch without
At least one stretch,
c) SEQ ID NO: 98;
d) An amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 98, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (as said affinity is measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference the amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; An amino acid residue stretch, which shall have about the same or higher potency (as defined herein);
e) an amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 121, said amino acid sequence comprising said amino acid residue stretch Has the same, nearly the same, or a higher affinity compared to an amino acid sequence comprising the amino acid residue stretch without three, two or one amino acid difference (the affinity is measured by surface plasmon resonance An amino acid sequence that binds to protein F of hRSV and / or contains an amino acid residue stretch that does not have three, two or one amino acid difference Amino acids that have the same, about the same, or higher potency (as defined herein) as compared to the sequence At least two (preferably identical) or at least three (preferably identical) amino acid sequences or Nanobodies selected from amino acid sequences comprising two or more amino acid residue stretches selected from no acid residue stretches (Registered Trademark) or a multivalent, preferably divalent or trivalent polypeptide comprising or consisting essentially of a stretch of amino acid residues corresponding to one of a) and b). As in the amino acid sequence forming part of the multivalent polypeptide, wherein the second stretch of amino acid residues is selected from one of c), d), e) and f). It is something.

Preferred multivalent (eg, divalent or trivalent) polypeptides have a first amino acid residue stretch,
a) SEQ ID NO: 98,
b) An amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 98, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (such as measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference an amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Consisting of stretches of amino acid residues, which should have about the same or higher potency (as defined herein) It is selected from,
The second amino acid residue stretch is
c) SEQ ID NO: 102,
d) a stretch of amino acid residues having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 102,
i) the amino acid residue stretch has aspartic acid (Asp, D) at position 6 (position 54 determined by Kabat numbering),
ii) the amino acid sequence comprising the amino acid residue stretch has the same, substantially the same or higher affinity as compared to the amino acid sequence comprising the amino acid residue stretch having no three, two or one amino acid difference ( The affinity is as measured by surface plasmon resonance) and binds to protein F of hRSV and / or the amino acid sequence comprising the amino acid residue stretch has three, two or one amino acid differences. Selected from the group consisting of amino acid residue stretches that have the same, approximately the same, or higher potency (as defined herein) as compared to an amino acid sequence comprising the above amino acid residue stretches without And
The third amino acid residue stretch is
e) an amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 121, said amino acid sequence comprising said amino acid residue stretch Has the same, nearly the same, or a higher affinity compared to an amino acid sequence comprising the amino acid residue stretch without three, two or one amino acid difference (the affinity is measured by surface plasmon resonance An amino acid sequence that binds to protein F of hRSV and / or contains an amino acid residue stretch that does not have three, two or one amino acid difference Amino acids that have the same, about the same, or higher potency (as defined herein) as compared to the sequence At least two (preferably identical) or at least three (preferably identical) amino acids selected from an amino acid sequence comprising three or more amino acid residue stretches selected from the group consisting of noic acid residue stretches It may comprise or consist essentially of a sequence or a Nanobody®.

A multivalent (eg, divalent or trivalent) polypeptide consists of at least two framework regions (FR1 to FR4, respectively) and at least two complementarity determining regions (CDR1 to CDR3, respectively). May comprise or consist essentially of one (preferably identical) or at least three (preferably identical) amino acid sequences or Nanobodies®, wherein CDR2 comprises
a) SEQ ID NO: 102,
b) a stretch of amino acid residues having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 102,
i) the amino acid residue stretch has aspartic acid (Asp, D) at position 6 (position 54 determined by Kabat numbering),
ii) the amino acid sequence comprising the amino acid residue stretch has the same, substantially the same or higher affinity as compared to the amino acid sequence comprising the amino acid residue stretch having no three, two or one amino acid difference ( The affinity is as measured by surface plasmon resonance) and binds to protein F of hRSV and / or the amino acid sequence comprising the amino acid residue stretch has three, two or one amino acid differences. Selected from amino acid residue stretches that have the same, approximately the same, or higher potency (as defined herein) as compared to an amino acid sequence that includes the above amino acid residue stretches having no.

A multivalent (eg, divalent or trivalent) polypeptide consists of at least two framework regions (FR1 to FR4, respectively) and at least two complementarity determining regions (CDR1 to CDR3, respectively). May comprise or consist essentially of one (preferably identical) or at least three (preferably identical) amino acid sequences or Nanobodies®, wherein CDR2 comprises
a) a stretch of amino acid residues having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 102, or b) SEQ ID NO: 102;
i) the amino acid residue stretch has aspartic acid (Asp, D) at position 6 (position 54 determined by Kabat numbering),
ii) the amino acid sequence comprising the amino acid residue stretch has the same, substantially the same or higher affinity as compared to the amino acid sequence comprising the amino acid residue stretch having no three, two or one amino acid difference ( The affinity is as measured by surface plasmon resonance) and binds to protein F of hRSV and / or the amino acid sequence comprising the amino acid residue stretch has three, two or one amino acid differences. Selected from the group consisting of amino acid residue stretches that have the same, approximately the same, or higher potency (as defined herein) as compared to an amino acid sequence comprising the above amino acid residue stretches without And
At least one of CDR1 or CDR3 is
c) SEQ ID NO: 98;
d) An amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 98, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (such as measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference an amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Stretches of amino acid residues, or shall have about the same or higher potency (as defined herein) CDR1 selected from the group consisting of: and / or e) SEQ ID NO: 121, or f) an amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 121. The amino acid sequence comprising the stretch of amino acid residues has the same, substantially the same or higher affinity compared to the amino acid sequence comprising the stretch of amino acid residues having no three, two or one amino acid difference ( The affinity is as measured by surface plasmon resonance) and binds to protein F of hRSV and / or the amino acid sequence comprising the amino acid residue stretch has three, two or one amino acid differences. The same, about the same, or higher potency compared to an amino acid sequence comprising the amino acid residue stretch without CDR3 selected from the group consisting of amino acid residue stretches (as defined herein).
Is selected from

Preferably, the multivalent (eg, divalent or trivalent) polypeptide consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively) May comprise or consist essentially of at least two (preferably identical) or at least three (preferably identical) amino acid sequences or Nanobodies®,
CDR1 is
a) SEQ ID NO: 98, b) an amino acid residue stretch having only 3, preferably 2, more preferably only 1 amino acid difference from SEQ ID NO: 98, wherein the amino acid sequence comprising said amino acid residue stretch is The same, about the same, or a higher affinity compared to an amino acid sequence comprising said amino acid residue stretch without three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance An amino acid sequence that binds to protein F of hRSV and / or has an amino acid residue stretch that does not have three, two or one amino acid difference An amino acid residue that has the same, about the same, or higher potency (as defined herein) as compared to Selected from the group consisting of stretches,
CDR2 is
c) a stretch of amino acid residues having no more than 3, preferably no more than 2, and more preferably only 1 amino acid difference from SEQ ID NO: 102, or d)
i) the amino acid residue stretch has aspartic acid (Asp, D) at position 6 (position 54 determined by Kabat numbering),
ii) the amino acid sequence comprising the amino acid residue stretch has the same, substantially the same or higher affinity as compared to the amino acid sequence comprising the amino acid residue stretch having no three, two or one amino acid difference ( The affinity is as measured by surface plasmon resonance) and binds to protein F of hRSV and / or the amino acid sequence comprising the amino acid residue stretch has three, two or one amino acid differences. Selected from the group consisting of amino acid residue stretches that have the same, approximately the same, or higher potency (as defined herein) as compared to an amino acid sequence comprising the above amino acid residue stretches without And
CDR3 is
e) SEQ ID NO: 121,
f) an amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 121, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (such as measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference an amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Stretches of amino acid residues which should be of about the same or higher potency (as defined herein). It is selected from the group.

  In certain aspects, the multivalent (eg, divalent or trivalent) polypeptide comprises at least two (preferably identical) SEQ ID NO: 102, preferably SEQ ID NO: 98, SEQ ID NO: 102 and SEQ ID NO: 121. ) Or at least three (preferably identical) amino acid sequences or Nanobodies®.

In another aspect, the invention provides:
a) SEQ ID NO: 60 to SEQ ID NO: 76
b) an amino acid sequence having only one amino acid difference from one of SEQ ID NO: 60 to SEQ ID NO: 76, preferably only two, more preferably only one;
i) the amino acid sequence has glutamine (Gln, Q) at position 105 (the position is determined by Kabat numbering);
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) Comprising or consisting essentially of at least two (preferably identical) amino acid sequences or Nanobodies® selected from amino acid sequences (as defined herein). Polyvalent, preferably bivalent or trivalent, polypeptides are provided.

  In another preferred embodiment, the polypeptide of the invention comprises or consists essentially of at least two identical amino acid sequences or Nanobodies® selected from the group consisting of SEQ ID NO: 60-SEQ ID NO: 76. Become.

In another aspect, the invention provides:
a) SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76
b) only three, preferably only two and more preferably only one amino acid difference from one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75 and SEQ ID NO: 76 An amino acid sequence having
i) the amino acid sequence has glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and / or arginine (Arg, R) at position 83 (the above positions are determined by Kabat numbering; Desired),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) Comprising or consisting essentially of at least two (preferably identical) amino acid sequences or Nanobodies® selected from amino acid sequences (as defined herein). Polyvalent, preferably bivalent or trivalent, polypeptides are provided.

  In another preferred aspect, the polypeptide of the invention comprises at least two identical polypeptides selected from the group consisting of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76. Comprises or consists essentially of an amino acid sequence or Nanobody®.

In another preferred aspect, the invention provides a method comprising:
a) SEQ ID NO: 65 and SEQ ID NO: 76,
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NO: 65 and SEQ ID NO: 76,
i) the amino acid sequence is aspartic acid (Asp, D) at position 54, glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and / or arginine (Arg, R) at position 83; (The position is determined by Kabat numbering),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) Comprising or consisting essentially of at least two (preferably identical) amino acid sequences or Nanobodies® selected from amino acid sequences (as defined herein). Polyvalent, preferably bivalent or trivalent, polypeptides are provided.

  In another preferred aspect, the polypeptide of the invention comprises or consists essentially of at least two identical amino acid sequences or Nanobodies® selected from the group consisting of SEQ ID NO: 65 and SEQ ID NO: 76. Become.

In another preferred aspect, the invention provides a method comprising:
a) SEQ ID NO: 60 to SEQ ID NO: 76
b) an amino acid sequence having only one amino acid difference from one of SEQ ID NO: 60 to SEQ ID NO: 76, preferably only two, more preferably only one;
i) the amino acid sequence has glutamine (Gln, Q) at position 105 (the position is determined by Kabat numbering);
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) Comprising or consisting essentially of at least three (preferably identical) amino acid sequences or Nanobodies® selected from amino acid sequences (as defined herein). A multivalent, preferably trivalent, polypeptide is provided.

  In another preferred aspect, the polypeptide of the invention comprises or consists essentially of at least three identical amino acid sequences or Nanobodies® selected from the group consisting of SEQ ID NO: 60-SEQ ID NO: 76. Become.

In another preferred aspect, the invention provides a method comprising:
a) SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76
b) only three, preferably only two and more preferably only one amino acid difference from one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75 and SEQ ID NO: 76 An amino acid sequence having
i) the amino acid sequence has glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and / or arginine (Arg, R) at position 83 (the above positions are determined by Kabat numbering; Desired),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) Comprising or consisting essentially of at least three (preferably identical) amino acid sequences or Nanobodies® selected from amino acid sequences (as defined herein). A multivalent, preferably trivalent, polypeptide is provided.

  In another preferred aspect, the polypeptide of the invention has at least three identical amino acid sequences selected from SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76, or Includes or consists essentially of Nanobodies®.

In another preferred aspect, the invention provides a method comprising:
a) SEQ ID NO: 65 and SEQ ID NO: 76,
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NO: 65 and SEQ ID NO: 76,
i) the amino acid sequence is aspartic acid (Asp, D) at position 54, glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and / or arginine (Arg, R) at position 83; (The position is determined by Kabat numbering),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) Comprising or consisting essentially of at least three (preferably identical) amino acid sequences or Nanobodies® selected from amino acid sequences (as defined herein). A multivalent, preferably trivalent, polypeptide is provided.

  In another preferred embodiment, the polypeptide of the invention comprises or consists essentially of at least three identical amino acid sequences or Nanobodies® selected from the group consisting of SEQ ID NO: 65 and SEQ ID NO: 76. Become.

  Preferred multivalent polypeptides of the invention comprise or consist essentially of three amino acid sequences having SEQ ID NO: 62 or Nanobodies®. Another preferred multivalent polypeptide of the invention comprises or consists essentially of three amino acid sequences having SEQ ID NO: 65 or Nanobodies®. Another preferred multivalent polypeptide of the invention comprises or consists essentially of three amino acid sequences having SEQ ID NO: 76 or Nanobodies®.

In another aspect, the invention provides:
a) SEQ ID NO: 146 to SEQ ID NO: 153
b) an amino acid sequence having only one, preferably two, more preferably only one amino acid difference from one of SEQ ID NOs: 146 to 153,
i) the amino acid sequence has proline (Pro, P) at position 14, arginine (Arg, R) at position 19, leucine (Leu, L) at position 20, and leucine (Leu, L) at position 108 (The above position is determined by Kabat numbering),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) Comprising or consisting essentially of at least two (preferably identical) amino acid sequences or Nanobodies® selected from amino acid sequences (as defined herein). Polyvalent, preferably bivalent or trivalent, polypeptides are provided.

  In another preferred embodiment, the polypeptide of the invention comprises or consists essentially of at least two identical amino acid sequences or Nanobodies® selected from the group consisting of SEQ ID NO: 146 to SEQ ID NO: 153. Become.

In another aspect, the present invention provides: a) SEQ ID NOs: 146-149 and SEQ ID NOs: 151-153;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153,
i) the amino acid sequence is
SEQ ID NO: 146 (this amino acid sequence preferably has glutamine (Gln, Q) at position 105);
SEQ ID NO: 147 (this amino acid sequence preferably has arginine (Arg, R) at position 83 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 148 (this amino acid sequence preferably has glutamic acid (Glu, E) at position 85 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 149 (this amino acid sequence preferably has arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 151 (this amino acid sequence preferably has arginine (Arg, R) at position 83);
SEQ ID NO: 152 (this amino acid sequence preferably has glutamic acid (Glu, E) at position 85);
SEQ ID NO: 153 (this amino acid sequence preferably has arginine (Arg, R) at position 83 and glutamic acid (Glu, E) at position 85)
The amino acid sequence has a proline (Pro, P) at position 14 and an arginine (Arg, R) at position 19, as in the case of having only 3, preferably 2, and more preferably only 1 amino acid difference. Leucine (Leu, L) at position 20, Leucine (Leu, L) at position 108, arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85, and / or glutamine at position 105 (Gln, Q) (the position is determined by Kabat numbering),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) Comprising or consisting essentially of at least two (preferably identical) amino acid sequences or Nanobodies® selected from amino acid sequences (as defined herein). Polyvalent, preferably bivalent or trivalent, polypeptides are provided.

  In another preferred embodiment, the polypeptide of the invention has at least two identical amino acid sequences or Nanobodies® selected from the group consisting of SEQ ID NOs: 146-149 and SEQ ID NOs: 151-153. Include or consist essentially of.

In another preferred aspect, the invention provides a method comprising:
a) SEQ ID NO: 146 to SEQ ID NO: 153
b) an amino acid sequence having only one, preferably two, more preferably only one amino acid difference from one of SEQ ID NOs: 146 to 153,
i) the amino acid sequence has proline (Pro, P) at position 14, arginine (Arg, R) at position 19, leucine (Leu, L) at position 20, and leucine (Leu, L) at position 108 (The above position is determined by Kabat numbering),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) Comprising or consisting essentially of at least three (preferably identical) amino acid sequences or Nanobodies® selected from amino acid sequences (as defined herein). A multivalent, preferably trivalent, polypeptide is provided.

  In another preferred embodiment, the polypeptide of the invention comprises or consists essentially of at least three identical amino acid sequences or Nanobodies® selected from the group consisting of SEQ ID NO: 146-SEQ ID NO: 153. Become.

In another preferred aspect, the invention provides a method comprising:
a) SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153,
i) the amino acid sequence is
SEQ ID NO: 146 (this amino acid sequence preferably has glutamine (Gln, Q) at position 105);
SEQ ID NO: 147 (this amino acid sequence preferably has arginine (Arg, R) at position 83 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 148 (this amino acid sequence preferably has glutamic acid (Glu, E) at position 85 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 149 (this amino acid sequence preferably has arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 151 (this amino acid sequence preferably has arginine (Arg, R) at position 83);
SEQ ID NO: 152 (this amino acid sequence preferably has glutamic acid (Glu, E) at position 85);
SEQ ID NO: 153 (this amino acid sequence preferably has arginine (Arg, R) at position 83 and glutamic acid (Glu, E) at position 85)
The amino acid sequence has a proline (Pro, P) at position 14 and an arginine (Arg, R) at position 19, as in the case of having only 3, preferably 2, and more preferably only 1 amino acid difference. Leucine (Leu, L) at position 20, Leucine (Leu, L) at position 108, arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85, and / or glutamine at position 105 (Gln, Q) (the position is determined by Kabat numbering),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) Comprising or consisting essentially of at least three (preferably identical) amino acid sequences or Nanobodies® selected from amino acid sequences (as defined herein). A multivalent, preferably trivalent, polypeptide is provided.

  In another preferred embodiment, the polypeptide of the invention has at least three identical amino acid sequences or Nanobodies® selected from the group consisting of SEQ ID NOs: 146-149 and SEQ ID NOs: 151-153. Include or consist essentially of.

In another aspect, the invention provides:
a) SEQ ID NO: 138 to SEQ ID NO: 141 and SEQ ID NO: 154 to SEQ ID NO: 157;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 138 to 141 and 154 to 157,
i) the amino acid sequence has aspartic acid (Asp, D) at position 1 (the position is determined by Kabat numbering);
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) A polyvalent, preferably bivalent, preferably comprising or consisting essentially of at least one amino acid sequence or Nanobody® selected from amino acid sequences (as defined herein). A multivalent or trivalent polypeptide is provided.

  In another preferred embodiment, does the polypeptide of the invention comprise at least one amino acid sequence or Nanobody® selected from the group consisting of SEQ ID NO: 138 to SEQ ID NO: 141 and SEQ ID NO: 154 to SEQ ID NO: 157? Or consisting essentially of:

In another preferred aspect, the invention provides a method comprising:
a) SEQ ID NO: 138 to SEQ ID NO: 141 and SEQ ID NO: 154 to SEQ ID NO: 157;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 138 to 141 and 154 to 157,
i) the amino acid sequence has aspartic acid (Asp, D) at position 1 (the position is determined by Kabat numbering);
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) (As defined herein) comprising, or consisting essentially of, at least one amino acid sequence or Nanobody® selected from an amino acid sequence. Provided is a multivalent polypeptide.

  In another preferred embodiment, the polypeptide of the invention comprises or consists essentially of at least one amino acid sequence or Nanobody selected from the group consisting of SEQ ID NO: 138 to SEQ ID NO: 141 and SEQ ID NO: 154 to SEQ ID NO: 157. Trivalent polypeptide.

In another preferred aspect, the invention provides the following polypeptide:
a) SEQ ID NO: 77 to SEQ ID NO: 79 and SEQ ID NO: 158;
b) a polypeptide which has no more than 3, preferably no more than 2, and more preferably no more than one amino acid difference from one of SEQ ID NOs: 77-79 and 158,
i) the amino acid sequence or Nanobody (registered trademark) contained in the polypeptide is glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, arginine (Arg, R) at position 83; And / or has glutamic acid (Glu, E) at position 85 (the position is determined by Kabat numbering),
ii) the polypeptide has the same, about the same, or a higher affinity as compared to a polypeptide that does not have three, two or one amino acid difference (as the affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or wherein said polypeptide has the same, about the same, or higher potency (as compared to a polypeptide without three, two or one amino acid difference) (As defined herein) is provided.

  Preferred trivalent polypeptides of the invention comprise or consist essentially of one of SEQ ID NOs: 77-79 and SEQ ID NO: 158.

In another preferred aspect, the invention provides the following polypeptide:
a) SEQ ID NO: 78 and SEQ ID NO: 79,
b) a polypeptide having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NO: 78 and SEQ ID NO: 79,
i) The amino acid sequence or Nanobody (registered trademark) contained in the polypeptide is aspartic acid (Asp, D) at position 54, glutamine (Gln, Q) at position 105, and leucine (Leu, L) at position 78 Having arginine (Arg, R) at position 83 and / or glutamic acid (Glu, E) at position 85 (the position is determined by Kabat numbering);
ii) the polypeptide has the same, about the same, or a higher affinity as compared to a polypeptide that does not have three, two or one amino acid difference (as the affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or wherein said polypeptide has the same, about the same, or higher potency (as compared to a polypeptide without three, two or one amino acid difference) (As defined herein) is provided.

  Preferred trivalent polypeptides of the invention comprise or consist essentially of SEQ ID NO: 78 and SEQ ID NO: 79.

  In another particular aspect, a polypeptide of the invention consists essentially of the amino acid sequence of SEQ ID NO: 77. In another particular aspect, a polypeptide of the invention consists essentially of the amino acid sequence of SEQ ID NO: 78. In another particular aspect, a polypeptide of the invention consists essentially of the amino acid sequence of SEQ ID NO: 79. In another particular aspect, a polypeptide of the invention consists essentially of the amino acid sequence of SEQ ID NO: 158.

In another preferred aspect, the invention provides the following polypeptide:
a) SEQ ID NO: 159 to SEQ ID NO: 161,
b) a polypeptide having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 159 to 161;
i) the amino acid sequence or Nanobody (registered trademark) contained in the polypeptide is proline (Pro, P) at position 14, arginine (Arg, R) at position 19, leucine (Leu, L) at position 20, And leucine (Leu, L) at position 108 (the position is determined by Kabat numbering),
iii) the polypeptide has the same, about the same, or a higher affinity as compared to the polypeptide without three, two or one amino acid difference (as the affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said polypeptide has the same, about the same, or higher potency (as compared to a polypeptide without three, two or one amino acid difference) (As defined herein) is provided.

  Preferred trivalent polypeptides of the invention comprise or consist essentially of one of SEQ ID NOs: 159-161.

In another preferred aspect, the invention provides the following polypeptide:
a) SEQ ID NO: 159 to SEQ ID NO: 161,
b) a polypeptide having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 159 to 161;
i) the polypeptide is
SEQ ID NO: 159 (amino acid sequence or Nanobody® included in the polypeptide preferably has arginine (Arg, R) at position 83 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 160 (the amino acid sequence or Nanobody® included in the above polypeptide is preferably arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85 and glutamine (Gln, Q)),
SEQ ID NO: 161 (The amino acid sequence or Nanobody® included in the polypeptide preferably has arginine (Arg, R) at position 83 and glutamic acid (Glu, E) at position 85)
And the amino acid sequence or Nanobody (R) encompassed within the polypeptide, as in the case of having only 3, preferably 2, and more preferably only 1 amino acid difference. , P), arginine (Arg, R) at position 19, leucine (Leu, L) at position 20, leucine (Leu, L) at position 108, arginine (Arg, R) at position 83, and glutamic acid at position 85 (Glu, E), and / or glutamine (Gln, Q) at position 105 (the position is determined by Kabat numbering),
ii) the polypeptide has the same, about the same, or a higher affinity as compared to an amino acid sequence that does not have three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or wherein said polypeptide has the same, approximately the same, or higher potency (as compared to an amino acid sequence having no three, two or one amino acid difference). (As defined herein) is provided.

  Preferred trivalent polypeptides of the invention comprise or consist essentially of SEQ ID NO: 159 to SEQ ID NO: 161.

In another preferred aspect, the invention provides the following polypeptide:
a) SEQ ID NOs: 142-145 and SEQ ID NOs: 162-165;
b) a polypeptide having no more than 3, preferably no more than 2, and more preferably no more than one amino acid difference from one of SEQ ID NOs: 142-145 and SEQ ID NOs: 162-165,
i) the first amino acid sequence or Nanobody® included in the polypeptide has aspartic acid (Asp, D) at position 1 (the position is determined by Kabat numbering);
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) (As defined herein) is provided.

  Preferred trivalent polypeptides of the invention comprise or consist essentially of SEQ ID NOs: 142-145 and SEQ ID NOs: 162-165.

Polypeptides having these sequences, for example, improved stability, reduced immunogenicity, (as further described herein (actual or apparent) K _D values (actual or apparent) K _A Value, k _on rate and / or k _off rate, or alternatively measured and / or expressed as an IC ₅₀ value), improved hRSV affinity for protein F and / or It exhibits beneficial properties for use as prophylactic, therapeutic and / or pharmacologically active agents, such as improved binding activity and / or improved efficacy and / or efficacy in neutralizing hRSV.

  The invention further comprises or consists essentially of one or more of the amino acid sequences, Nanobodies® and / or polypeptides (or suitable fragments thereof) of the invention, optionally one or more further. It relates to compounds or constructs, in particular proteins or polypeptides (also referred to herein as "compound (s) of the invention"), comprising a plurality of other groups, residues, moieties or linking units. As will be apparent to those skilled in the art from the further disclosure herein, such additional groups, residues, moieties, binding units or amino acid sequences may be incorporated into an amino acid sequence, Nanobody® or polypeptide of the invention. (And / or the compound or construct in which it is present) may or may not be provided with additional functional groups, and may alter the properties of the amino acid sequence, Nanobody® and / or polypeptide of the invention. Or it may not be changed.

Use of portions, fragments, analogs, mutants, variants, alleles and / or derivatives of the amino acid sequences and / or polypeptides of the invention, and / or one or more such portions, fragments, analogs The use of proteins or polypeptides that comprise or consist essentially of the forms, mutants, variants, alleles and / or derivatives is also contemplated as long as they are suitable for the uses envisioned herein. Within the scope of the invention. Such portions, fragments, analogs, mutants, variants, alleles and / or derivatives are usually (at least part of) a functional antigen binding site for binding of hRSV to antigen site II on protein F. containing, more preferably is capable of specifically binding an antigen site II on protein F of hRSV, as described further more herein preferably ((actual or apparent) K _D value (actual or apparent) _{K a value k on} rate and / or _{k off} rate, or alternatively is preferably measured as _{IC 50} values, and / or represented by) protein F of hRSV affinity It is possible to bind to the above antigen site II. Such portions, fragments, analogs, mutants, variants, alleles and / or derivatives typically also have the efficacy and / or efficacy of hRSV neutralization as defined herein. Some non-limiting examples of such parts, fragments, analogs, mutants, variants, alleles, derivatives, proteins and / or polypeptides will become clear from the further description herein. Will. Further fragments or polypeptides of the invention may also be provided by suitably combining (ie, by ligation or gene fusion) one or more (smaller) portions or fragments as described herein. .

  The invention also relates to nucleic acid or nucleotide sequences encoding the amino acid sequences of the invention, Nanobodies® of the invention and / or polypeptides (or suitable fragments thereof) of the invention. Such nucleic acids are also referred to herein as "nucleic acid (s) of the invention" and may be in the form of a genetic construct as further described herein. Thus, the invention also relates to nucleic acid or nucleotide sequences in the form of a genetic construct.

  The invention further expresses (or can be expressed under suitable circumstances) the amino acid sequences of the invention, Nanobodies® of the invention, polypeptides of the invention and / or compounds or constructs of the invention. And / or a host or host cell containing the nucleic acid of the invention. Some preferred but non-limiting examples of such hosts or host cells will become clear from the further description herein.

  The present invention further provides at least one inventive amino acid sequence (or a suitable fragment thereof), at least one inventive Nanobody®, at least one inventive polypeptide, at least one inventive compound or The construct, at least one monovalent construct according to the invention, and / or at least one nucleic acid according to the invention, and optionally, ie, depending on the intended use of the composition, one or more of such compositions known per se or A product or composition containing or comprising a plurality of further components. Such a product or composition can be, for example, a pharmaceutical composition (as described herein) or a veterinary composition. Some preferred, but non-limiting examples of such products or compositions will become clear from the further description herein.

  The invention further relates to methods of preparing the amino acid sequences, Nanobodies®, polypeptides, nucleic acids, host cells, products and compositions described herein.

  The invention further relates to the application and use of the amino acid sequences, Nanobodies®, polypeptides, compounds, nucleic acids, host cells, products and compositions described herein, and to the respiratory track caused by hRSV. And b) a method for preventing and / or treating infectious diseases. Some preferred but non-limiting applications and uses will become apparent from the further description herein.

  The amino acid sequences, Nanobodies®, polypeptides, compounds and compositions of the present invention are generally used to block the interaction of hRSV protein F with target host cells and / or their membranes. to neutralize hRSV (various hRSV strains and / or escape variants), to modulate, inhibit and / or prevent hRSV infectivity (of various hRSV strains and / or escape variants); To regulate, inhibit and / or prevent the fusion of the target hRSV strain and / or escape mutant with the target host cell and / or (of various hRSV strains and / or escape mutants). ) Can be used to modulate, inhibit and / or prevent hRSV entry in a targeted host cell.

  Accordingly, the amino acid sequences, Nanobodies®, polypeptides, compounds and compositions of the present invention can be used for the prevention and / or treatment of diseases and disorders associated with hRSV infection. Examples of such diseases and disorders associated with hRSV infection will be apparent to those of skill in the art based on the disclosure herein, including the following diseases and disorders: respiratory diseases, upper respiratory tract infections, Respiratory tract infections, bronchiolitis (inflammation of the peripheral airways in the lungs), pneumonia, dyspnea, cough, (recurrent) wheezing and asthma.

  Accordingly, the present invention provides for respiratory illness, upper respiratory tract infection, lower respiratory tract infection, bronchiolitis (inflammation of the peripheral airways in the lungs), pneumonia, dyspnea, cough, (recurrent) wheezing and asthma caused by hRSV. A method of preventing and / or treating a pharmaceutically active amount of at least one amino acid sequence of the invention, a Nanobody® of the invention, a polypeptide of the invention, a compound or construct of the invention or It also relates to a method comprising administering a monovalent construct of the invention, or a composition of the invention, to a subject in need thereof.

  The present invention relates to the prevention and / or prevention of respiratory diseases, upper respiratory tract infections, lower respiratory tract infections, bronchiolitis (inflammation of the peripheral airways in the lungs), pneumonia, dyspnea, cough, (recurrent) wheezing and / or asthma. Or an amino acid sequence of the invention, a Nanobody® of the invention, a method of the invention for use in the preparation of a pharmaceutical composition for therapy and / or in one or more of the methods described herein. Or a compound or construct of the invention, or a monovalent construct of the invention.

  The present invention relates to the prevention and / or prevention of respiratory diseases, upper respiratory tract infections, lower respiratory tract infections, bronchiolitis (inflammation of the peripheral airways in the lungs), pneumonia, dyspnea, cough, (recurrent) wheezing and / or asthma. Or an amino acid sequence of the invention, a Nanobody® of the invention, a polypeptide of the invention, a compound or construct of the invention, or a monovalent construct of the invention for therapy.

  Other applications and uses of the amino acid sequences, Nanobodies®, polypeptides and compounds of the present invention, and compositions of the present invention will become apparent to those skilled in the art from the further disclosure herein.

Competition ELISA: Synagis (R) Fab is, is a diagram showing for binding to _{F TM-} protein, purified RSV binding Nanobodies (R) and competing with Numax Fab as described in Example 8 . Nanobody® 202A5 is an irrelevant Nanobody® that binds to influenza HA. FIG. 11 shows the binding of _FTM- protein to monovalent, divalent and trivalent Nanobodies® as described in Example 10. FIG. 11 shows the efficacy of the monovalent, divalent and trivalent constructs as described in Example 11 to neutralize Long and B-1 RSV strains (RSV neutralization). FIG. 11 shows the efficacy of the monovalent, divalent and trivalent constructs as described in Example 11 to neutralize Long and B-1 RSV strains (RSV neutralization). FIG. 4 shows the neutralization assay of RSV Long and escape mutants R7C2 / 1, R7C2 / 11 and R7.936 / 4 with monovalent Nanobody® 7B2 in the concentration range from about 2 μM to 6 nM. Curve fitting was performed only on the monovalent Nanobody® data. FIG. 4 shows the neutralization assay of RSV Long and the escape mutants R7C2 / 1, R7C2 / 11 and R7.936 / 4 with monovalent Nanobody® 15H8 in the concentration range from about 2 μM to 6 nM. Curve fitting was performed only on the monovalent Nanobody® data. FIG. 4 shows the neutralization assay of RSV Long and escape mutants R7C2 / 1, R7C2 / 11 and R7.936 / 4 by monovalent Nanobody® NC41 in the concentration range from about 2 μM to 6 nM. Curve fitting was performed only on the monovalent Nanobody® data. FIG. 4 shows the neutralization assay of RSV Long and escape mutants R7C2 / 1, R7C2 / 11 and R7.936 / 4 with Trivalent Nanobody® RSV400 in a concentration range of about 20 nM to 100 pM. FIG. 3 shows the neutralization assay of RSV Long and escape mutants R7C2 / 1, R7C2 / 11 and R7.936 / 4 with Trivalent Nanobody® RSV404 in a concentration range of about 20 nM to 100 pM. FIG. 3 shows the neutralization assays of RSV Long and escape mutants R7C2 / 1, R7C2 / 11 and R7.936 / 4 with Trivalent Nanobody® RSV407 in a concentration range of about 20 nM to 100 pM. FIG. 3 shows the neutralization assay of RSV Long and escape mutants R7C2 / 1, R7C2 / 11 and R7.936 / 4 with Trivalent Nanobody® RSV403 in a concentration range of about 20 nM to 100 pM. FIG. 11 shows neutralization of RSV Long strain and RSV B-1 strain by trivalent NC41 Nanobody (registered trademark) having different linker lengths as described in Example 15. FIG. 2 is a schematic diagram of humanized residues introduced into selected NC41 mutants. The dots indicate the presence of wild type residues and the letters correspond to humanized residues. Numbering shall follow Kabat numbering. FIG. 2 shows an alignment of a preferred humanized Nanobody® sequence of the invention. Continuation of FIG. 7A. FIG. 4 shows the neutralization of the hRSV Long strain and the B-1 strain by monovalent and trivalent humanized NC41 mutants. This figure compares the neutralization by the two trivalent humanized NC41 mutants (RSV414 and RSV426) to that of the corresponding monovalent Nanobody®. FIG. 4 shows the neutralization of the hRSV Long strain and the B-1 strain by monovalent and trivalent humanized NC41 mutants. This figure shows neutralization by trivalent NC41 mutants (RSV414, RSV426 and RSV427). FIG. 2 shows the neutralization of hRSV Long strain by RSV407 and RSV434. FIG. 4 shows the neutralization of the hRSV B-1 strain by RSV407 and RSV434. FIG. 14 shows a (preventive) efficacy study of RSV407 in a cotton rat model as described in Example 21. Twenty-four hours (day -1) prior to RSV infection (day 0), rats were intranasally administered various concentrations of RSV407. Virus titers (mean ± standard error, n = 6) were determined in lung lavages at peak levels of viremia on day 4 post-infection ( ^* = two-tailed Student's t test for untreated group). P <0.05). FIG. 14 shows a (preventive) efficacy study of RSV407 in a cotton rat model as described in Example 21. Twenty-four hours (day -1) prior to RSV infection (day 0), rats were intranasally administered various concentrations of RSV407. Viral RNA levels were determined in the lung lavage fluid at the peak level of viremia 4 days post infection ( ^* = P <0.05 in a two-tailed Student's t test for the untreated group). FIG. 21 shows a (therapeutic) efficacy study of RSV434 in a cotton rat model as described in Example 21. Rats were infected with RSV on day 0 and treated with 0 mg / kg, 1 mg / kg, 2 mg / kg, 4 mg / kg or 20 mg / kg RSV434 by intranasal instillation on days 2 and 3 (n = 6). Viral titers were determined in nasal washes at peak levels of viremia (4 days post infection). The horizontal line indicates the detection limit. FIG. 21 shows a (therapeutic) efficacy study of RSV434 in a cotton rat model as described in Example 21. Rats were infected with RSV on day 0 and treated with 0 mg / kg, 1 mg / kg, 2 mg / kg, 4 mg / kg or 20 mg / kg RSV434 by intranasal instillation on days 2 and 3 (n = 6). Virus titers were determined in lung lavage at peak levels of viremia (4 days post infection). The horizontal line indicates the detection limit.

In the description, examples and claims:
a) Unless otherwise indicated or defined, all terms used have their ordinary meaning in the art, which will be clear to the skilled person. Reference is made, for example, to the standard handbook mentioned in paragraph a) on page 46 of WO 08/020079.

b) Unless otherwise indicated, the terms "immunoglobulin sequence", "sequence", "nucleotide sequence" and "nucleic acid" are described in WO 08/020079 at page 46, paragraph b). It is like.

c) Unless otherwise indicated, all methods, steps, techniques and operations not specifically described can be carried out in a manner known per se, which will be clear to the skilled person. Also, for example, standard handbooks and the general background art referred to herein, as well as further references cited therein and, for example, the following review, Presta, Adv. Drug Deliv. Rev. 2006, 58 (5-6): 640-56, Levin and Weiss, Mol. Biosyst. 2006, 2 (1): 49-57, Irving et al., J. Immunol. Methods, 2001, 248 (1-2), 31. -45, Schmitz et al., Placenta, 2000, 21 Suppl. A, S106-12, Gonzaleset al., Tumour Biol., 2005, 26 (1), 31-43 (These are protein engineering techniques such as affinity maturation. And other techniques for improving the specificity and other desired properties of proteins such as immunoglobulins).

d) Amino acid residues are indicated according to the standard three letter amino acid code or one letter amino acid code. Reference is made to Table A-2 on page 48 of WO 08/020079.

e) When a nucleotide sequence or amino acid sequence is "comprising" or "consisting essentially of" another nucleotide or amino acid sequence, respectively, this means WO 08/020079. Has the meaning given in paragraph i) of pages 51-52.

f) The term "essentially in isolated form" has the meaning given in WO 08/020079, pages 52 and 53, paragraph j).

g) The terms "domain" and "binding domain" have the meaning given in WO 08/020079, page 53, paragraph k).

h) The terms "antigenic determinant" and "epitope" can also be used interchangeably herein and have the meaning given in l) paragraph 53 of WO 08/020079.

i) specific antigenic determinants, epitopes, antigens or proteins (or with respect to at least one part, fragment or epitope thereof) and (specifically, as further described in paragraph m) on page 53 of WO 08/020079. An amino acid sequence capable of binding, having affinity for a particular antigenic determinant, epitope, antigen or protein, and / or having specificity for a particular antigenic determinant, epitope, antigen or protein (eg, The Nanobodies®, antibodies, polypeptides, or generally antigen-binding proteins or polypeptides, or fragments thereof of the invention) may be directed against, or “against,” the antigenic determinant, epitope, antigen or protein. (Directed against).

j) The term "specificity" has the meaning given in paragraph n) of WO 08/020079 page 53 to page 56 and refers to a particular antigen binding molecule or protein as mentioned therein. (Eg, a Nanobody® or polypeptide of the invention) represents the number of different types of antigens or antigenic determinants to which a molecule can bind. The specificity of an antigen binding protein may be determined based on affinity and / or avidity based on WO 08/020079 (herein incorporated by reference) (herein an antigen binding molecule, such as a Nanobody® of the invention). ) Or polypeptides) and related techniques are also described. Some preferred techniques are also described) at pages 53-56. Typically, the antigen binding protein (e.g., the amino acid sequence, Nanobody (R) and / or polypeptide of the invention) comprises ^10-5 mol / L to ^10-12 mol / L or less, and preferably ^10-7 mol / L. mol / ^{L～10 -12} mol / L or less, and more preferably ^{10 -8} mol / ^L～10 at ^-12 mol / L dissociation constant _{(K D)} (i.e. ¹⁰ 5 L / mol ^{to 10 12} L / mole It binds to these antigens as described above, and preferably with a binding constant (K _A ) of at least 10 ⁷ L / mol to 10 ¹² L / mol, and more preferably of 10 ⁸ L / mol to 10 ¹² L / mol. 10 ⁴ mol / L greater than any _{K D} values (i.e. ¹⁰ 4 ^M -1 (any _{K A} value less than L / mol)) is generally considered to indicate nonspecific binding. Preferably, the monovalent immunoglobulin sequence of the invention binds to the desired antigen with an affinity of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM (eg 10 nM to 5 nM). Specific binding between an antigen binding protein and an antigen or antigenic determinant can be determined by any suitable method known per se (eg, Scatchard analysis and / or competitive binding assays (eg, radioimmunoassay (RIA), enzyme immunoassays). EIA) and sandwich competition assays) and various modifications thereof known per se in the art, as well as other techniques mentioned herein. As will be apparent to those skilled in the art, and as described in WO 08/020079 at pages 53-56, the dissociation constant may be the actual or apparent dissociation constant. Methods for determining dissociation constants will be apparent to those skilled in the art, and include, for example, the techniques mentioned on pages 53-56 of WO 08/020079.

k) The half-life of the amino acid sequences, compounds or polypeptides of the present invention can be generally defined as described in WO 08/020079, page 57, paragraph o), where as mentioned, for example, 50% reduction in serum concentration of amino acid sequence, compound or polypeptide in vivo due to degradation of amino acid sequence, compound or polypeptide by natural mechanism and / or elimination or sequestration of amino acid sequence, compound or polypeptide The time it takes to do. The in vivo half-life of the amino acid sequence, compound or polypeptide of the present invention can be determined by any method known per se (for example, pharmacokinetic analysis). Suitable techniques will be apparent to those skilled in the art, and may for example be as generally described in WO 08/020079, page 57, paragraph o). Also, as mentioned in paragraph o) of page 57 of WO 08/020079, the half-life utilizes parameters such as t1 / 2-α, t1 / 2-β and area under the curve (AUC). Can be expressed as See, for example, the following experimental parts, and standard handbooks (eg, Kenneth, A et al: Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and Peters et al, Pharmacokinetic analysis: A Practical Approach (1996)). See also "Pharmacokinetics", M Gibaldi & D Perron, published by Marcel Dekker, 2nd Rev. edition (1982). The term "increased half-life" or "increased half-life" refers in particular to an increase in t1 / 2-β, as also defined in paragraph o) on page 57 of WO 08/020079. Either or both of 2-α and / or AUC may or may not be increased.

l) With respect to a target or antigen, the term "interaction site" on the target or antigen refers to a site for binding to a receptor or other binding partner, a catalytic site, a cleavage site, a site for allosteric interaction, a site for a target or antigen. Sites of amino acid residues on the target or antigen, epitopes, antigenic determinants, portions, domains or stretches that are sites involved in multimerization (such as homomerization or heterodimerization), or organisms of the target or antigen Means any other site, epitope, antigenic determinant, moiety, domain or stretch of amino acid residues on a target or antigen involved in a biological action or mechanism. More generally, an "interaction site" refers to the amino acid sequence or polypeptide of the present invention wherein the amino acid sequence or polypeptide (as defined herein) is a target or antigen (and / or any pathway in which the target or antigen is involved). Any site, epitope, antigenic determinant, moiety, domain of an amino acid residue on a target or antigen that can bind to modulate, interaction, signaling, biological mechanism or action) Or it can be a stretch.

m) the amino acid sequence or polypeptide is at least 10-fold better, such as at least 100-fold, preferably at least 1000-fold, and up to 10,000-fold better than the affinity of said amino acid sequence or polypeptide for binding to a second target or antigen; Do affinity (as described above, preferably _the K _D value, K _a value, K _off expressed as a speed and / or K _on speed) if it binds the first antigen with, compared to the second target or antigen , Is "specific for" a first target or antigen. For example, the amino acid sequence or polypeptide is said amino acid sequence or polypeptide is at least 10 times smaller than the K _D for binding to a second target or antigen, such as at least 100 times less, and preferably at least 1000 times smaller, for example, 10000 in more than twice smaller K _D values may bind to the first target or antigen. Preferably, if the amino acid sequence or polypeptide is "specific" for the first target or antigen relative to the second target or antigen, then the amino acid sequence or polypeptide is defined as defined herein. As such) is directional to the first target or antigen, but not directional to the second target or antigen.

n) The term “(cross) -block”, “(cross) -blocked” and “(cross) -blocking”) is used interchangeably herein and refers to amino acid sequence or other It refers to the ability of a binding agent (eg, a polypeptide of the invention) to prevent binding of another amino acid sequence or binding agent of the invention to a given target. The extent to which the amino acid sequence or other binding agent of the invention can prevent the binding of another amino acid sequence or other binding agent to a target, and thus whether it can be said that it can be cross-blocked according to the present invention, competitive binding It can be determined using an assay. One particularly suitable quantitative cross-blocking assay uses a Biacore instrument that can measure the extent of interaction using surface plasmon resonance techniques. Another suitable quantitative cross-blocking assay uses an ELISA-based approach that measures competition between amino acid sequences or other binding agents for their binding to the target.

In general, the following describes a Biacore assay suitable for determining whether an amino acid sequence or other binding agent cross-blocks or is capable of cross-blocking according to the present invention. It will be appreciated that this assay can be used with any of the amino acid sequences or other binding agents described herein. Biacore equipment (eg, Biacore 3000) operates according to the manufacturer's recommendations. Thus, in one cross-blocking assay, the target protein is coupled to a CM5 Biacore chip using standard amine coupling chemistry to create a target-coated surface. Typically, 200 to 800 resonance units of the target are attached to the chip (an amount that gives an easily measurable level of binding, but can be easily saturated by the concentration of test reagent used). Two test amino acid sequences (designated A ^* and B ^* ), which evaluate the ability to cross block each other, are mixed in a suitable buffer at a 1: 1 binding site molar ratio to form a test mixture. When calculating binding site-based concentrations, the molecular weight of an amino acid sequence is considered to be the total molecular weight of the amino acid sequence divided by the number of target binding sites on this amino acid sequence. The concentration of each amino acid sequence in the test mixture should be high enough to easily saturate the binding site for this amino acid sequence on the target molecule captured on the Biacore chip. The amino acid sequences in the mixture are present at the same molar concentration (on a binding site basis), and the concentration is usually between 1.00 and 1.5 micromolar (on a binding site basis). Separate solutions containing A ^* and B ^* alone are also prepared. A ^* and B ^{* in} these solutions are assumed to be in the same buffer and at the same concentration as the test mixture. The test mixture is passed over a target-coated Biacore chip and the total binding recorded. The chip is then processed to remove the bound amino acid sequence without compromising the chip binding target. Typically, this is done by treating the chip with 30 mM HCl for 60 seconds. The solution of A ^* alone is then passed over the target-coated surface and the amount of binding recorded. In addition, the chip is processed to remove any bound amino acid sequence without compromising the chip binding target. A solution of B ^* alone is then passed over the target-coated surface and the amount of binding recorded. Next, the theoretical maximum binding of the mixture of A ^* and B ^* is calculated, which is the sum of the binding of each amino acid sequence when passed alone on the target surface. If the binding of the mixture actually recorded is less than this theoretical maximum, the two amino acid sequences cross-block each other. Thus, in general, an amino acid sequence or other binding agent according to the invention that cross-blocks the recorded binding combined during the assay and in the presence of a second amino acid sequence or other binding agent of the invention. 80% to 0.1% (eg, 80% to 4%) of the theoretical maximum binding (defined immediately above) of two amino acid sequences or binding factors, specifically 75% to 0.1% (eg, 75%) of the theoretical maximum binding % To 4%), and more specifically, 70% to 0.1% (eg, 70% to 4%) of the theoretical maximum binding to bind to the target in the Biacore cross-blocking assay described above. The Biacore assay described above is the primary assay used to determine whether amino acid sequences or other binding factors cross-block each other according to the present invention. In rare cases, a particular amino acid sequence or other binding agent may not bind to the target that is bound via the amine chemistry to the CM5 Biacore chip (usually the relevant binding site on the target is plugged by binding to the chip). This happens when it is destroyed or destroyed). In such a case, cross-blocking can be determined using a tagged, eg, N-terminal, His-tagged target. In this particular format, after binding the anti-His amino acid sequence to the Biacore chip, the His-tagged target is passed over the surface of the chip and captured with the anti-His amino acid sequence. After each chip regeneration cycle, a cross-blocking analysis is performed essentially as described above, except that the surface coated with the anti-His amino acid sequence is loaded back with a new His-labeled target. In addition to the examples given as using an N-terminal His-tagged target, a C-terminal His-tagged target can alternatively be used. In addition, various other labels and combinations of label binding proteins known in the art can be used for such cross-blocking analyses (eg, HA labeling with anti-HA antibody; FLAG labeling with anti-FLAG antibody; Strepto). Biotin labeling with avidin).

  In general, the following describes an ELISA assay for determining whether an amino acid sequence or other binding agent directed to a target cross-blocks or is capable of cross-blocking, as defined herein. ing. It will be appreciated that this assay can be used with any of the amino acid sequences described herein (or other binding agents, such as a polypeptide of the invention). The general principle of this assay is to have a target-directed amino acid sequence or binding agent coated on the wells of the ELISA plate. An excess of a second, potentially cross-blocking, anti-target amino acid sequence is added to the solution (ie, does not bind to the ELISA plate). Then, a limited amount of target is added to the wells. The coated amino acid sequence and the amino acid sequence in solution compete for the binding of a limited number of target molecules. The plate is washed to remove excess target that is not bound to the coated amino acid sequence, and also to the second solution phase amino acid sequence and any formed between the second solution phase amino acid sequence and the target. Remove the complex. Thereafter, the amount of bound target is measured using appropriate reagents to detect the target. An amino acid sequence in a solution that is capable of cross-blocking the coated amino acid sequence will result in a coating that is less than the number of target molecules that can bind to the coated amino acid sequence in the absence of the second solution phase amino acid sequence. The number of target molecules that can bind to the amino acid sequence can be reduced. When selecting a first amino acid sequence, eg, Ab-X, to immobilize the amino acid sequence, the first amino acid sequence is coated on the wells of an ELISA plate, and the plate is then blocked with a suitable blocking solution. Minimize non-specific binding of subsequently added reagents. An excess of the second, such that the number of moles of Ab-Y target binding site per well is at least 10 times the number of moles of Ab-X target binding site used per well used during coating of the ELISA plate. , Ie Ab-Y, is then added to the ELISA plate. The targets are then added such that the number of moles of target added per well per well is at least 25-fold less than the number of Ab-X target binding sites used to coat each well. After a suitable incubation period, the ELISA plate is washed, a target detection reagent is added, and the amount of target specifically bound to the coated anti-target amino acid sequence (in this case, Ab-X) is measured. The background signal for the assay was the coated amino acid sequence (Ab-X in this case), the amino acid sequence of the second solution phase (Ab-Y in this case), target buffer only (i.e. no target present) and target detection It is defined as the signal obtained in the well using the reagent. Positive control signals for the assay were: the coated amino acid sequence (Ab-X in this case), the second solution phase amino acid sequence buffer only (i.e., no second solution phase amino acid sequence was present), the target and the target detection reagent. Is defined as the signal obtained in the well. An ELISA assay can be performed such that the positive control signal is at least six times the background signal. Any artifacts resulting from the choice of which amino acid sequence to use as the coating amino acid sequence and which amino acid sequence to use as the second (competitive) amino acid sequence (eg, significantly different Ab-X and Ab-Y against the target) In order to avoid affinity), the cross-blocking assay can be performed in two formats: 1) Format 1 is where the Ab-X is the amino acid sequence coating on the ELISA plate and the Ab-Y is the solution 2) Format 2 is the amino acid sequence to be coated on the ELISA plate, and Ab-X is the competitive amino acid sequence in the solution. Is the case. Ab-X and Ab-Y were used in either Format 1 or Format 2 where the solution phase anti-target amino acid sequence was the target detection signal (ie, positive control) obtained in the absence of the solution phase anti-target amino acid sequence. 60% to 100%, specifically 70% to 100%, and more specifically 80% to 100% reduction of the target detection signal (ie, the amount of target bound by the coated amino acid sequence) compared to the Is defined as cross-blocking if it can cause

o) The amino acid sequence is identical to that of protein F from various strains of hRSV, such as protein F from various strains of hRSV, such as protein F from various different mammalian species such as human serum albumin and cynomolgus monkey serum albumin. "Specific" (as defined herein) for both two different antigens or antigenic determinants are "cross-reactive" for both of these different antigens or antigenic determinants. There is.

p) As further described herein, the total number of amino acid residues in the Nanobody® can be in the range of 110-120, preferably 112-115, and most preferably 113. However, portions, fragments, analogs or derivatives of Nanobodies® (as further described herein) meet the additional requirements outlined herein, and preferably It should be noted that there is no particular limitation on its length and / or size as long as it is suitable for the purposes described in.

q) As further described in paragraphs q) on pages 58 and 59 of WO 08/020079 (incorporated herein by reference), the amino acid residues of Nanobodies® were obtained from Riechmann and Muyldermans, J. Immunol Methods 2000 Jun 23 ; 240 (1-2):. 185-195 papers (see for example Figure 2 of this article) to be applied to V _HH domains from camelids in , According to the general numbering for V _H domains given by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91) (Registered trademark) contains amino acid residues 1 to 30; Nanobody (registered trademark) CDR1 contains amino acid residues 31 to 35; and Nanobody (registered trademark) FR2 has 36 amino acid residues. The amino acid residues at positions 49 to 49, the CDR2 of Nanobody® includes amino acid residues at positions 50 to 65, the FR3 of Nanobody® includes the amino acid residues at positions 66 to 94, The CDR3 of Nanobody® contains amino acid residues at positions 95 to 102, and the FR4 of Nanobody® contains amino acid residues 103 to 113.

r) In the context of the present invention, "target host cell (of a virus)" generally refers to a specific cell that is or is derived from a living subject susceptible to infection by the virus.

s) The term "viral infectivity" as used herein, refers to the percentage of living subjects that become infected with the virus when exposed to the virus.

t) As used herein, the term "virus neutralization" refers to any suitable in vitro, cellular or in vivo assay (such as those referred to herein) (eg, Anderson et al. 1985). Microneutralization assays described by (J. Clin. Microbiol. 22: 1050-1052) and 1988 (J. Virol. 62: 4232-4238), for example, variants of these assays as described in Example 6; For example, measured using a plaque reduction assay as described by Johnson et al. 1997 (J. Inf. Dis. 176: 1215-1224, and variations thereof and those referred to herein). And the modulation and / or reduction and / or prevention and / or inhibition of the infectivity (as defined herein) of the virus by binding of the neutralizing compound to the virion. In particular, "neutralizing (a virus)" or "to neutralize (a virus)" is a suitable in vitro, cellular or in vivo assay (as referred to herein). The normal (ie, native) of the virus in the same assay under the same conditions but without the amino acid sequence, Nanobody® or polypeptide of the invention, as measured using ) The infectivity (as defined herein) of the virus is at least 1%, preferably at least 5%, such as at least 10% or at least 25%, as compared to the infectivity (as defined herein). %, Such as at least 50%, at least 60%, at least 70%, at least 80%, or more than 90% of any of the modulating, reducing, preventing or inhibiting. It may mean.

u) The terms "potency of the amino acid sequence of the present invention", "potency of the Nanobody (registered trademark) of the present invention", "potency of the polypeptide of the present invention", and / or "potency of the compound or construct of the present invention" As used herein, is a specific virus (e.g., such as the Nanobody® of the invention, the polypeptide of the invention, and / or the compound or construct of the invention, as described above, hRSV), regulate, inhibit and / or prevent infectivity of the virus, regulate, inhibit and / or prevent fusion of the virus with (the cell membrane of) the target host cell, and / or It refers to the ability to modulate, inhibit and / or prevent entry of a virus (as defined herein) into a targeted host cell. This potency may be determined by any suitable assay known in the art or described herein, such as the microneutralization assay as described in the Examples section and / or the assays mentioned in item t) above. Can be measured.

v) As used herein, the term “viral attachment” refers to either directly (eg, by interaction with a viral receptor) or indirect (eg, with one or more other proteins or molecules). Attachment of a virus (eg, hRSV) to a targeted host cell (by mediating interaction with a viral receptor).

w) The term "viral fusion," as used herein, can be direct (e.g., by interaction with a membrane compound of the targeted host cell) or indirect (e.g., one or more other Fusion of a virus (eg, hRSV) with a target host cell (by mediating the interaction of a target protein or molecule with a membrane compound of the target host cell).

x) The term "viral entry" as used herein is any that is required to achieve virion attachment to and / or viral fusion with the targeted host cell. Virus-mediated biological pathways.

y) “Amino acid residue stretch” means two or more amino acid residues that are adjacent to each other or in close proximity to each other (ie, in the primary or tertiary structure of the amino acid sequence). In the context of the present invention, "amino acid residue stretch" refers to the binding (at least partially) of the amino acid sequence, Nanobody®, polypeptide, compound or construct of the present invention to antigenic site II on protein F of hRSV. To) get involved.

z) When comparing two amino acid residue stretches (or two CDR sequences), the term "amino acid difference" refers to a), c) or e) amino acid residue stretches (or CDR sequences), A single amino acid residue insertion, deletion or substitution at a certain position in the amino acid residue stretch (or CDR sequence) specified in b), d) or f), respectively. The stretch of amino acid residues (or CDR sequences) of b), d) and f) is one, two or at most three such stretches as compared to the stretch of amino acid residues of a), c) or e), respectively. It is understood that various amino acid differences can be included.

  "Amino acid difference" refers to any one, two, or both of the amino acid sequence of the invention that improves the properties of the amino acid sequence or that does not at least unduly compromise the desired property or the balance or combination of the desired properties of the amino acid sequence of the invention. There may be one or up to three substitutions, deletions and / or insertions, or any combination thereof. In this regard, the resulting amino acid sequence of the invention is compared to an amino acid sequence comprising at least one, two or at most three, one or more amino acid residue stretches without substitutions, deletions and / or insertions. Amino acids of the invention that bind to protein F of hRSV with the same, approximately the same or higher affinity (the affinity is as measured by surface plasmon resonance) and / or the resulting amino acids of the invention The sequence is at least the same, about the same, or higher compared to an amino acid sequence comprising one or more amino acid residue stretches without one, two, or up to three substitutions, deletions and / or insertions Shall have effect. Those skilled in the art will generally recognize, based on the disclosure herein, optionally introducing, for example, a limited number of possible substitutions, deletions or insertions, and the effect on the properties of the amino acid sequence thus obtained. After a limited degree of routine experimentation, which may involve the determination, a suitable substitution, deletion and / or insertion, or a suitable combination thereof, could be determined and selected.

  For example, depending on the host organism used to express the amino acid sequences of the present invention, and within the capabilities of those skilled in the art, such deletions and / or substitutions may be made by one or more post-translational modifications. It can be designed to remove sites (eg, one or more glycosylation sites).

  In a preferred embodiment of the invention, "amino acid differences" are amino acid substitutions. The amino acid substitution improves any of the properties of the amino acid sequence of the present invention, or at least one, two or any of the desired properties of the amino acid sequence of the present invention or at least does not unduly impair the balance or combination of the desired properties. There can be up to three substitutions. In this regard, the resulting amino acid sequence of the invention is at least the same, about the same, or at least as compared to an amino acid sequence comprising one or more amino acid residue stretches without one, two or at most three substitutions. It shall bind to protein F of hRSV with higher affinity (as said affinity is measured by surface plasmon resonance) and / or obtain at least one amino acid sequence of the invention, Shall have the same, approximately the same, or higher potency as compared to an amino acid sequence comprising one or more amino acid residue stretches without two or at most three substitutions, deletions and / or insertions.

  The person skilled in the art will generally be able to optionally introduce, for example, a limited number of possible substitutions, based on the disclosure herein, and determine the effect on the properties of the Nanobody® thus obtained. After a limited degree of routine experimentation, a suitable substitution could be sought and selected.

  Amino acid substitutions in one or more amino acid residue stretches can be conservative amino acid substitutions. “Conservative” amino acid substitutions generally involve the replacement of an amino acid residue by another amino acid residue of similar chemical structure, and little or essential effect on the function, activity or other biological property of the resulting amino acid sequence. Amino acid substitution that has no effect on Such conservative amino acid substitutions are described, for example, in WO 04/037999, GB 3357768, WO 98/49185, WO 00/46383 and WO 01/09300. (Preferred) types and / or combinations of such substitutions are known from WO 04/037999 and WO 98/49185, and further references mentioned therein. Can be selected based on relevant teachings from.

  Such conservative substitutions are preferably those in which one amino acid in the following groups (a) to (e) is replaced with another amino acid residue in the same group: Aliphatic non-polar or slightly polar residues of the molecule: Ala, Ser, Thr, Pro and Gly, (b) polar negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln, (c) polar and positively charged residues: His, Arg and Lys, (d) giant aliphatic non-polar residues: Met, Leu, Ile, Val and Cys, and (e) aromatic Residues: Phe, Tyr and Trp.

  Particularly preferred conservative substitutions are as follows: Ala to Gly or Ser, Arg to Lys, Asn to Gln or His, Asp to Glu, Cys to Ser, Gln to Asn, Glu to Asp, Gly to Ala or Pro, His to Asn or Gln, Ile to Leu or Val, Leu to Ile or Val, Lys to Arg, Gln or Glu, Met to Leu To Tyr or Ile, Phe to Met, Leu or Tyr, Ser to Thr, Thr to Ser, Trp to Tyr, Tyr to Trp, and / or Phe to Val, Ile or Leu.

  Amino acid substitutions in one or more amino acid residue stretches may provide amino acid sequences with increased affinity for binding of hRSV to protein F. This may be achieved by techniques such as random mutagenesis or site-directed mutagenesis, and / or for example WO 09/004065, WO 05/003345, WO 06/023144, European Patent No. This can be done by other techniques per se known for affinity maturation, such as described in EP 527 809, EP 398 834 and the like.

Without limitation, the rules (partially or completely obeying) for the substitution of amino acid residues in CDRs may be as follows (ie, substitution with an amino acid having similar side chain chemistry):
Replacing K with R;
R is replaced by K,
A is replaced with S or T;
Replacing S with A or T;
Replacing T with A or S;
Replacing I with L or V;
Replacing L with I or V;
Replacing V with I or L;
Replace F with Y,
Replace Y with F,
Replace N with D,
Replace D with N,
Replace Q with E,
Replace E with Q,
Replace G with A,
Replace M with L and keep H, C, W and P constant.

In addition, but not limited to, the rules (partially or completely obeying) for the substitution of amino acid residues in CDRs may alternatively be 27-35 and 50-58 (Kabat numbering system With respect to the substitution used) may be as follows:
For positions 27-35,
The original amino acid residue at position 27 (using Kabat numbering) is F; G; R; S; two of F, G, R, S; and three of F, G, R, S Or all of these, preferably all of them,
A; I; S; T; two of A, I, S, and T; three of A, I, S, and T for the original amino acid residue at position 28 (using Kabat numbering) Or all of these, preferably all of them,
F: G; L; S; two of F, G, L, S; three of F, G, L, S, the original amino acid residue at position 29 (using Kabat numbering) Or all of these, preferably all of them,
The original amino acid residue at position 30 (using Kabat numbering) is D; G; S; T; two of D, G, S, T; and three of D, G, S, T Or all of these, preferably all of them,
The original amino acid residue at position 31 (using Kabat numbering) is D; I; N; S; T; two of D, I, N, S, T; D, I, N, S, Three of T; or all of these, preferably all of them,
Substituting the original amino acid residue at position 32 (using Kabat numbering) with D; N; Y; two of D, N, Y; or all, preferably all of them;
A: G; T; V; two of A, G, T, and V; three of A, G, T, and V represent the original amino acid residue at position 33 (using Kabat numbering). Or all of these, preferably all of them,
Substituting the original amino acid residue at position 34 (using Kabat numbering) with I; M; or all, preferably all of them;
Substituting the original amino acid residue at position 35 (using Kabat numbering) with A; G; S; two of A, G, S; or all, preferably all of them;
If the original amino acid sequence has an amino acid sequence at position 52a (using Kabat numbering), for positions 50-58,
A: C; G; S; T; two of A, C, G, S, T; A, C, G, S, 50 (using Kabat numbering) Three of T; four of A, C, G, S, T; or all, preferably all of them,
Substituting the original amino acid residue at position 51 (using Kabat numbering) with I,
The original amino acid residue at position 52 (using Kabat numbering) is N; R; S; T; two of N, R, S, T; and three of N, R, S, T Or all of these, preferably all of them,
The original amino acid residue at position 52a (using Kabat numbering) is R; S; T; W; two of R, S, T, and W; three of R, S, T, and W Or all of these, preferably all of them,
The original amino acid residue at position 53 (using Kabat numbering) is D; G; N; S; T; two of D, G, N, S, T; D, G, N, S, Three of T; four of D, G, N, S, T; or all, preferably all of these,
Substituting the original amino acid residue at position 54 (using Kabat numbering) with D; G; or all, preferably all of them;
Substituting the original amino acid residue at position 55 (using Kabat numbering) with D; G; S; two of D, G, S; or all, preferably all of them;
The original amino acid residue at position 56 (using Kabat numbering) is I; N; R; S; T; two of I, N, R, S, T; I, N, R, S, Three of T; four of I, N, R, S, T; or all, preferably all of these,
Substituting the original amino acid residue at position 57 (using Kabat numbering) with T,
The original amino acid residue at position 58 (using Kabat numbering) is D; H; N; S; Y; two of D, H, N, S, Y; Three of Y; four of D, H, N, S, Y; or all, preferably all of these,
If the original amino acid sequence has no amino acid sequence at position 52a (using Kabat numbering), for positions 50-58,
A; G; R; S; T; two of A, G, R, S, T; A, G, R, S, 50 (using Kabat numbering) Three of T; four of A, G, R, S, T; or all, preferably all of these,
Substituting the original amino acid residue at position 51 (using Kabat numbering) with I,
Substituting the original amino acid residue at position 52 (using Kabat numbering) with N; S; T; two of N, S, T; or all, preferably all of them;
The original amino acid residue at position 53 (using Kabat numbering) is N; R; S; T; Y; two of N, R, S, T, Y; Three of Y; four of N, R, S, T, Y; or all, preferably all of them,
The original amino acid residue at position 54 (using Kabat numbering) is D; G; R; S; two of D, G, R, S; and three of D, G, R, S Or all of these, preferably all of them,
Substituting the original amino acid residue at position 55 (using Kabat numbering) with G,
The original amino acid residue at position 56 (using Kabat numbering) is G; N; R; S; T; two of D, N, R, S, T; D, N, R, S, Three of T; four of D, N, R, S, T; or all, preferably all of these,
Substituting the original amino acid residue at position 57 (using Kabat numbering) with T,
D; N; T; Y; two of D, N, T, and Y; three of D, N, T, and Y, using the original amino acid residue at position 58 (using Kabat numbering). Or all of these, preferably all of them,
One or more of the potentially useful substitutions (or combinations thereof) thus determined (in any manner known per se, as further described herein) may be substituted by the CDR sequence can be introduced into the affinity for the protein F of hRSV, and / or such (as further described herein (actual or apparent) K _D values (actual or apparent) K _a values, k _on rate and / or _{k off} rate, or alternatively is preferably measured as _{IC 50} values, and / or represented by) improved binding characteristics, improved affinity for a protein F of hRSV and / or avidity The resulting amino acid sequence (s) can be tested for other desired properties, such as improved and / or improved efficacy and / or efficacy of neutralizing hRSV. In this manner, one of ordinary skill in the art can determine other suitable substitutions in CDRs (or suitable combinations thereof) by limited trial and error based on the disclosure herein.

  Amino acid sequences comprising stretches of amino acid residues having one, two or at most three substitutions, insertions or deletions, and the nucleic acid sequences encoding them, can be obtained in any manner known per se, for example in WO It can be provided using one or more of the techniques mentioned on pages 103 and 104 of 08/020079.

Amino acid sequence is preferably the present invention obtained, as defined herein (as further described herein (actual or apparent) K _D values (actual or apparent) K _A values, bind to protein F of hRSV with an affinity (suitably measured and / or expressed as a k _on rate and / or a k _off rate, or alternatively an IC ₅₀ value) and / or HRSV shall be neutralized with efficacy and / or efficacy as specified herein.

aa) When comparing two amino acid sequences, the term "amino acid difference" refers to the insertion, deletion of a single amino acid residue at a position in a first amino acid sequence relative to a second amino acid sequence. Or represents substitution. It is understood that two amino acid sequences may contain one, two, or up to three such amino acid differences.

  An "amino acid difference" is in an amino acid sequence that improves the properties of the amino acid sequence of the invention or at least does not unduly impair the desired properties of the amino acid sequence of the invention or the balance or combination of the desired properties, i.e., Any in one or more framework regions or in one or more CDRs (which may be a CDR of the invention (ie, CDR2) or another CDR (ie, CDR1, CDR2 or CDR3)), or any combination thereof One, any two or at most three substitutions, deletions or insertions. In this regard, the resulting amino acid sequence of the present invention has at least the same, approximately the same or higher affinity compared to an amino acid sequence without at least one, two or at most three substitutions, deletions or insertions (see above). Affinity is as measured by surface plasmon resonance) and binds to protein F of hRSV and / or the resulting amino acid sequence of the invention comprises at least one, two or at most 3 Shall have the same, about the same, or higher potency as compared to an amino acid sequence having no substitutions, deletions and / or insertions. Those skilled in the art will generally recognize, based on the disclosure herein, optionally introducing, for example, a limited number of possible substitutions, deletions or insertions, and the effect on the properties of the amino acid sequence thus obtained. After a limited degree of routine experimentation, which may involve the determination, a suitable substitution, deletion or insertion, or a suitable combination thereof, could be determined and selected.

  In one aspect of the invention, an "amino acid difference" is an amino acid substitution. The amino acid substitutions improve one or more framework regions that do not improve the properties of the amino acid sequence of the invention or at least unduly compromise the desired properties or the balance or combination of the desired properties of the amino acid sequences of the invention. Or any one or more of the CDRs (which may be CDRs of the invention (ie, CDR2) or another CDR (ie, CDR1, CDR2 or CDR3)), or any one, two or more of the CDRs in any combination thereof. May be three substitutions. In this regard, the resulting amino acid sequence of the invention has at least the same, substantially the same or higher affinity compared to an amino acid sequence without at least one, two or at most three substitutions (where the affinity is a surface plasmon (As determined by resonance) and / or the resulting amino acid sequence of the invention has at least one, two or at most three substitutions Shall have the same, substantially the same, or higher potency as compared to the amino acid sequence. One skilled in the art may generally include, for example, introducing a limited number of possible substitutions, and determining the effect on the properties of the amino acid sequence thus obtained, based on the disclosure herein. After a limited degree of routine experimentation, a suitable substitution could be sought and selected.

  As noted above, substitutions, insertions or deletions may be in one or more framework regions and / or in one or more CDRs. As described above (see item z above), an amino acid substitution in one or more CDRs is any substitution, such as a "conservative substitution" (as defined herein). May also work according to certain rules (as defined herein) and / or may lead to improved properties on the resulting amino acid sequence.

When such substitutions, insertions or deletions are made in one or more framework regions, the substitutions, insertions or deletions may be made (e.g., WO 08/020079, Tables A-3 to A-8). May be performed at one or more characteristic residues and / or at one or more other positions in the framework residues (as defined in Substitutions, insertions or deletions at characteristic residues (unless such suitable humanized substitutions) are generally less preferred. By way of non-limiting example, the substitution may be, for example, a conservative substitution (as described herein) and / or another amino acid residue that is naturally occurring at the same position in another _VHH domain. Although it may be replaced by an amino acid residue (see WO 08/020079, Tables A-5 to A-8), the invention is not generally limited thereto.

Substitutions, insertions or deletions made (preferably) in one or more framework regions are humanized substitutions (i.e., one or more amino acids in the amino acid sequence of the native _VHH sequence, especially in the framework sequence). The residues may be replaced by one or more amino acid residues occurring at the corresponding position (s) in the _VH domain of a conventional human four-chain antibody). Some preferred but non-limiting humanized substitutions (and suitable combinations thereof) will be apparent to those of skill in the art based on the disclosure herein. By comparing the sequence of one of the framework regions of the amino acid sequences of the invention as defined in a) with the corresponding framework sequence of one or more closely related human _VH sequences, Identified one or more potentially useful humanized substitutions (or combinations thereof), which are then known per se (as further described herein). HRSV may be introduced into the amino acid sequence of the invention as defined above under a), wherein hRSV has an affinity for protein F, stability, ease and level of expression, and / or The resulting humanized amino acid sequence can be tested for other desired properties defined. In this manner, one skilled in the art can determine other suitable humanized substitutions (or suitable combinations thereof) by limited trial and error based on the disclosure herein.

  The humanized substitution should be selected such that the resulting humanized amino acid sequence and / or Nanobody® still retains the advantageous properties of Nanobody® as defined herein. . One of skill in the art will generally be able to build upon the disclosure herein and optionally, for example, to introduce a limited number of possible humanized substitutions, and to characterize the Nanobodies® thus obtained. After a limited degree of routine experimentation that could involve determining its effects, a suitable humanized substitution or a combination of appropriate humanized substitutions could be determined and selected.

In general, as a result of humanization, the amino acid sequences and / or Nanobodies® of the invention still retain the advantageous properties of Nanobodies® of the invention as described herein, It can be more “human-like”. As a result, such humanized amino acid sequences and / or Nanobodies® may have several advantages, such as reduced immunogenicity as compared to the corresponding naturally occurring _VHH domain. Similarly, the skilled artisan will appreciate, on the basis of the disclosure herein, and after any limited degree of routine experimentation, on the one hand, the advantageous properties afforded by the humanized substitutions, and, on the other hand, the natural _VHH domain A humanized substitution or combination of suitable humanized substitutions could be sought and selected that would optimize or achieve the desired or appropriate balance between advantageous properties.

  The amino acid sequence and / or Nanobody® of the present invention may comprise any framework residue (s), such as one or more characteristic residues (as defined herein) or one or more. Can be suitably humanized with other framework residues (ie, non-characteristic residues) or any suitable combination thereof. One of the preferred humanized substitutions for Nanobodies® of the “P, R, S-103 group” or “KERE group” (as defined in WO 08/020079) is from Q108 to L108. Is a substitution of

  Depending on the host organism used to express the amino acid sequence, Nanobody® or polypeptide of the present invention, such deletions and / or substitutions may be made post-translationally within the skill of the art. One or more sites for modification (eg, one or more glycosylation sites) can also be designed to be removed. Alternatively, substitutions or insertions (as described herein) may introduce one or more binding sites for a functional group, eg, site-specific (as also described herein). It can be designed to allow PEGylation.

As is evident from the V _HH entropy and V _HH variability data shown in Tables A-5 to A-8 of WO 08/020079, some amino acid residues within the framework regions are more likely than others. High preservability. In general, any substitutions, deletions or insertions will preferably take place at positions that are not easily conserved, but the invention is not limited in this broadest sense. Also, in general, amino acid substitutions are preferred over amino acid deletions or insertions.

Any amino acid substitutions applied to the polypeptides described herein may also be made by analyzing the frequency of amino acid mutations between homologous proteins of different species, developed by Schulz et al., Principles of Protein Structure, Springer-Verlag, 1978. Analysis of structure forming potentials developed by Chou and Fasman, Biochemistry 13: 211, 1974 and Adv. Enzymol., 47: 45-149, 1978, and Eisenberg et al., Proc. Natl. Acad Sci. USA 81: 140-144, 1984; developed by Kyte &Doolittle; J. Molec. Biol. 157: 105-132, 1981, and Goldman et al., Ann. Rev. Biophys. Chem. 15: 321-353, 1986. Based on the analysis of the hydrophobic patterns in the proteins (all of which are incorporated herein by reference in their entirety). Information on the primary, secondary and tertiary structure of Nanobodies® is given in the description herein and in the general background art referred to above. Since this, the crystal structure of the V _HH domain from Lama example Desmyter et al., Nature Structural Biology , 3, 9, 803 (1996), Spinelli et al., Natural StructuralBiology (1996), 3, 752-757, And Decanniere et al., Structure, Vol. 7, 4, 361 (1999). More information on some amino acid residues that form V _H / V _L interfaces and potential camelizing substitutions at these positions in conventional V _H domains can be found in the prior art referred to above. .

  Amino acid sequences and / or Nanobodies® having one, two or at most three substitutions, insertions or deletions, and the nucleic acid sequences encoding them, can be obtained in any manner known per se, for example internationally. It can be provided using one or more of the techniques mentioned on pages 103 and 104 of WO 08/020079.

Amino acid sequence and / or Nanobody of the invention (R) is preferably obtained, as defined herein (as further described herein (actual or apparent) K _D value, (in fact (Or an apparent) K _A value, k _on rate and / or k _off rate, or alternatively, which is suitably measured and / or expressed as an IC ₅₀ value) that binds to protein F of hRSV with an affinity) And / or neutralize hRSV with efficacy and / or efficacy as defined herein.

bb) When comparing two polypeptides, the term "amino acid difference" refers to the insertion, deletion or insertion of a single amino acid residue at a position in a first polypeptide relative to a second polypeptide. Represents substitution. It is understood that two polypeptides may contain one, two, or up to three such amino acid differences.

An "amino acid difference" is in a polypeptide that improves the properties of the polypeptide of the invention, or at least unduly impairs the desired properties of the polypeptide of the invention or the balance or combination of the desired properties, i.e., Any in one or more framework regions or in one or more CDRs (which may be a CDR of the invention (ie, CDR2) or another CDR (ie, CDR1, CDR2 or CDR3)), or any combination thereof One, any two or up to three substitutions, deletions or insertions. In this regard, the resulting polypeptide of the invention has the same, about the same, or a higher affinity (as described above) as compared to a polypeptide without at least one, two or at most three substitutions, deletions or insertions. Affinity is as measured by surface plasmon resonance) and binds to the protein F of hRSV and / or the resulting polypeptide of the invention has at least one, two, or at most 3 Shall have the same, about the same, or a higher potency as compared to a polypeptide without two substitutions, deletions and / or insertions. Polypeptide is preferably obtained, as defined herein (as further described herein (actual or apparent) K _D values (actual or apparent) K _A value k _on rate and / or k _off rate, or defined by the alternatively is suitably measured as IC ₅₀ values, and / or represented) and those that bind to proteins F of hRSV in affinity, and / or described herein HRSV should be neutralized with such efficacy and / or efficacy. One of skill in the art will generally recognize, based on the disclosure herein, optionally introducing, for example, a limited number of possible substitutions, deletions or insertions, and the effect on the properties of the polypeptide thus obtained. After a limited degree of routine experimentation, which may involve the determination, a suitable substitution, deletion or insertion, or a suitable combination thereof, could be determined and selected.

In one aspect of the invention, an "amino acid difference" is an amino acid substitution. The amino acid substitutions improve one or more properties of the polypeptide of the invention or at least unduly impair the desired property or the balance or combination of the desired properties of the polypeptide of the invention, one or more framework regions. Or any one or more of the CDRs (which may be CDRs of the invention (ie, CDR2) or another CDR (ie, CDR1, CDR2 or CDR3)), or any one, any two in any combination thereof. Or up to any three substitutions. In this regard, the resulting polypeptide of the invention has the same, about the same, or a higher affinity (as described above) as compared to a polypeptide without at least one, two or at most three substitutions, deletions or insertions. Affinity is as measured by surface plasmon resonance) and binds to the protein F of hRSV and / or the resulting polypeptide of the invention has at least one, two, or at most 3 Shall have the same, about the same, or a higher potency as compared to a polypeptide without two substitutions, deletions and / or insertions. Polypeptide is preferably obtained, as defined herein (as further described herein (actual or apparent) K _D values (actual or apparent) K _A value k _on rate and / or k _off rate, or defined by the alternatively is suitably measured as IC ₅₀ values, and / or represented) and those that bind to proteins F of hRSV in affinity, and / or described herein HRSV should be neutralized with such efficacy and / or efficacy. One skilled in the art may generally include, for example, introducing a limited number of possible substitutions, and determining the effect on the properties of the polypeptide thus obtained, based on the disclosure herein. After a limited degree of routine experimentation, a suitable substitution could be sought and selected.

  As noted above, substitutions, insertions or deletions may be in one or more framework regions and / or in one or more CDRs. As noted above (see item z)), substitutions, insertions or deletions in the CDR may be made (as defined herein) by any possible substitutions, insertions or deletions, such as "conservative substitutions". Loss may be effected by certain rules (as defined herein) and / or may lead to improved properties for the resulting polypeptide.

When such substitutions, insertions or deletions are made in one or more framework regions, the substitutions, insertions or deletions may be made (e.g., WO 08/020079, Tables A-3 to A-8). May be performed at one or more characteristic residues and / or at one or more other positions in the framework residues (as defined in Substitutions, insertions or deletions at characteristic residues (unless such suitable humanized substitutions) are generally less preferred. By way of non-limiting example, the substitution may be, for example, a conservative substitution (as described herein) and / or another amino acid residue that is naturally occurring at the same position in another _VHH domain. Although it may be replaced by an amino acid residue (see WO 08/020079, Tables A-5 to A-8), the invention is not generally limited thereto.

Substitutions, insertions or deletions made (preferably) in one or more framework regions can be humanized substitutions. Some preferred but non-limiting humanized substitutions (and suitable combinations thereof) will be apparent to those of skill in the art based on the disclosure herein. comparing the sequence of one framework region of the amino acid sequence encompassed by the polypeptide of the invention as defined in a) with the corresponding framework sequence of one or more closely related human _VH sequences Identify potentially useful humanized substitutions, and then replace one or more of the thus determined potentially useful humanized substitutions (or combinations thereof) (as further described herein). HRSV can be introduced into the polypeptides of the invention as defined above under a) in any manner known per se, including the affinity of hRSV for protein F, stability, ease and level of expression, and And / or the resulting polypeptide sequence can be tested for other desired properties as defined herein. In this manner, one skilled in the art can determine other suitable humanized substitutions (or suitable combinations thereof) by limited trial and error based on the disclosure herein.

  The humanized substitutions should be chosen such that the resulting humanized polypeptide sequence still retains the advantageous properties of Nanobodies® included in the polypeptide as defined herein. One skilled in the art will generally be able to build, based on the disclosure herein, and optionally, eg, introduce a limited number of possible humanized substitutions, and include Nanobodies (eg, After a limited degree of routine experimentation, which may involve determining its effect on the properties of the RTM, a suitable humanized substitution or combination of appropriate humanized substitutions could be determined and selected.

In general, as a result of humanization, the polypeptides of the invention will retain the advantageous properties of the Nanobodies® of the invention encompassed by the polypeptides as described herein, while at the same time retaining more “ Human-like ". As a result, such a humanized polypeptide may have several advantages, for example, reduced immunogenicity as compared to a polypeptide that includes the corresponding naturally occurring _VHH domain. Similarly, the skilled artisan will appreciate, on the basis of the disclosure herein, and after any limited degree of routine experimentation, on the one hand, the advantageous properties afforded by the humanized substitutions, and, on the other hand, the natural _VHH domain A humanized substitution or a combination of suitable humanized substitutions could be selected which optimizes or achieves the desired or appropriate balance between advantageous properties.

  The polypeptides of the present invention may comprise any framework residue (s), such as one or more characteristic residues (as defined herein) or one or more other framework residues (as defined herein). (I.e., non-characteristic residues) or any suitable combination thereof. One suitable humanized substitution for Nanobodies® of the “P, R, S-103 group” or “KERE group” is a Q108 to L108 substitution.

  Depending on the host organism used to express the polypeptide of the invention, and within the skill of the art, such deletions and / or substitutions may be made at one or more sites for post-translational modifications (eg, For example, one or more glycosylation sites) can be designed. Alternatively, substitutions or insertions (as described herein) may introduce one or more binding sites for a functional group, eg, a site-specific PEG (as also described herein). Can be designed to allow for

As is evident from the V _HH entropy and V _HH variability data shown in Tables A-5 to A-8 of WO 08/020079, some amino acid residues within the framework regions are more likely than others. High preservability. In general, any substitutions, deletions or insertions will preferably take place at positions that are not easily conserved, but the invention is not limited in this broadest sense. Also, in general, amino acid substitutions are preferred over amino acid deletions or insertions.

Any amino acid substitutions applied to the polypeptides described herein may also be made by analyzing the frequency of amino acid mutations between homologous proteins of different species, developed by Schulz et al., Principles of Protein Structure, Springer-Verlag, 1978. , Chou and Fasman, Biochemistry 13: 211, 1974 and Adv.Enzymol., 47: 45-149, 1978.Analysis of the ability to form structures, and Eisenberg et al., Proc. Natl. Acad Sci. USA 81: 140-144, 1984, Kyte and Doolittle; J. Molec.Biol. 157: 105-132, 1981, and Goldman et al., Ann. Rev. Biophys. Chem. 15: 321-353, 1986. (All of which are incorporated herein by reference in their entirety). Information on the primary, secondary and tertiary structure of Nanobodies® is given in the description herein and in the general background art referred to above. Since this, the crystal structure of the V _HH domain from Lama example Desmyter et al., Nature Structural Biology , Vol.3, 9, 803 (1996), Spinelli et al., Natural StructuralBiology (1996), 3, 752- 757, and Decanniere et al., Structure, Vol. 7, 4, 361 (1999). More information on some amino acid residues that form V _H / V _L interfaces and potential camelizing substitutions at these positions in conventional V _H domains can be found in the prior art referred to above. .

  Polypeptides having one, two or at most three substitutions, insertions or deletions, and the nucleic acid sequences coding therefor, can be obtained in any manner known per se, for example WO 103/00079 Pages and 104 can be provided using one or more of the techniques mentioned.

Polypeptides are preferably the present invention obtained, as defined herein (as further described herein (actual or apparent) K _D values (actual or apparent) K _A values, bind to protein F of hRSV with an affinity (suitably measured and / or expressed as a k _on rate and / or a k _off rate, or alternatively an IC ₅₀ value) and / or HRSV shall be neutralized with efficacy and / or efficacy as specified herein.

cc) The drawings, sequence listing and experimental parts / examples are provided only to further illustrate the invention, and unless otherwise expressly indicated herein, the scope of the invention and / or the appended claims. It should in no way be construed as limiting the scope of the claims.

  Due to the binding of hRSV to its epitope on protein F, the amino acid sequence is usually composed of one or more amino acid residues or one or more amino acid residue stretches within the amino acid sequence (further described herein). As defined, ie, each “stretch” contains two or more amino acid residues that are adjacent to or adjacent to each other (ie, in the primary or tertiary structure of the amino acid sequence). Through this, the amino acid sequence of the present invention can bind to an epitope on protein F of hRSV. Thus, these amino acid residues or stretches of amino acid residues are referred to as “sites” that bind to an epitope on protein F of hRSV (also referred to herein as “antigen binding sites”, as further defined herein). ).

  The present invention provides a number of amino acid residue stretches (as defined herein) that are particularly suitable for binding to antigenic site II on protein F of hRSV (as described herein for the antigenic site in the hRSV F protein). See Lopez et al. 1998, J. virol. 72: 6922-6928). These stretches of amino acid residues may be present in and / or incorporated into an amino acid sequence of the invention, particularly to form (part of) the antigen binding site of the amino acid sequence of the invention. The resulting amino acid sequence is at antigen site II on protein F of hRSV, forms part of antigen site II on protein F of hRSV, or overlaps with antigen site II on protein F of hRSV (ie, primary Binds to a particular epitope on protein F of hRSV that is in close proximity to antigenic site II on protein F of hRSV (ie, in primary or tertiary structure). The resulting amino acid sequence of the invention may also be such that it can compete with Synagis® for binding to hRSV protein F, and / or the same as synagis® on hRSV protein F. Preferably, it is capable of binding to an epitope or binding site, or to an epitope that is close to and / or overlaps the binding site.

The present invention provides a stretch of amino acid residues (SEQ ID NO: 102) that is particularly suitable for binding to protein F of hRSV. This stretch of amino acid residues (or a variant of SEQ ID NO: 102 as defined herein) may be added to the amino acid sequence of the invention to form, in particular, (a portion of) the antigen binding site of the amino acid sequence of the invention. It may be present and / or incorporated therein. Amino acid residue stretches were generated against protein F of hRSV and further modified in a library approach to produce humanized NC41 Nanobodies® (as described in the Examples section), It is generated as: (SEQ ID NO: 5 NC41) CDR2 sequence or _{V HH} sequence of the antibody. More specifically, the glycine (Gly, G) residue at position 6 of CDR2 of NC41 was replaced with an aspartic acid (Asp, D) residue. Surprisingly, the amino acid sequence comprising the amino acid residue stretch (SEQ ID NO: 102), (further, as described herein (actual or apparent) K _D values (actual or apparent) K _A Value, k _on rate and / or k _off rate, or alternatively measured and / or expressed as an IC ₅₀ value), improved hRSV affinity for protein F and / or Improved binding activity and / or improved efficacy and / or efficacy in neutralizing hRSV was observed. This stretch of amino acid residues (or the variant of SEQ ID NO: 102 as defined herein) is also referred to herein as "the CDR2 sequence of the invention".

Thus, in one aspect, the invention provides:
a) SEQ ID NO: 102,
b) a stretch of amino acid residues having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 102,
i) the amino acid residue stretch has aspartic acid (Asp, D) at position 6 (position 54 determined by Kabat numbering),
ii) the amino acid sequence comprising the amino acid residue stretch has the same, substantially the same or higher affinity as compared to the amino acid sequence comprising the amino acid residue stretch having no three, two or one amino acid difference ( The affinity is as measured by surface plasmon resonance) and binds to protein F of hRSV and / or the amino acid sequence comprising the amino acid residue stretch has three, two or one amino acid differences. An amino acid selected from an amino acid residue stretch that has the same, approximately the same, or a higher potency (as defined herein) as compared to an amino acid sequence comprising the above amino acid residue stretch without An amino acid sequence comprising at least one residue stretch is provided.

In a preferred embodiment, the present invention provides an amino acid sequence comprising two or more stretches of amino acid residues, wherein one stretch comprises:
a) SEQ ID NO: 102,
b) a stretch of amino acid residues having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 102,
i) the amino acid residue stretch has aspartic acid (Asp, D) at position 6 (position 54 determined by Kabat numbering),
ii) the amino acid sequence comprising the amino acid residue stretch has the same, substantially the same or higher affinity as compared to the amino acid sequence comprising the amino acid residue stretch having no three, two or one amino acid difference ( The affinity is as measured by surface plasmon resonance) and binds to protein F of hRSV and / or the amino acid sequence comprising said stretch of amino acid residues differs by three, two or one amino acid Is selected from amino acid residue stretches that have the same, approximately the same, or higher potency (as defined herein) as compared to the amino acid sequence comprising the amino acid residue stretch without
At least one stretch,
c) SEQ ID NO: 98;
d) An amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 98, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (as said affinity is measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference the amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; An amino acid residue stretch, which shall have about the same or higher potency (as defined herein);
e) an amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 121, said amino acid sequence comprising said amino acid residue stretch Has the same, nearly the same, or a higher affinity compared to an amino acid sequence comprising the amino acid residue stretch without three, two or one amino acid difference (the affinity is measured by surface plasmon resonance An amino acid sequence that binds to protein F of hRSV and / or contains an amino acid residue stretch that does not have three, two or one amino acid difference Amino acids that have the same, about the same, or higher potency (as defined herein) as compared to the sequence The amino acid residue stretch selected from the amino acid residue stretch and corresponding to one of a) and b) is such that it is always present in the amino acid sequence of the invention, and the second amino acid residue stretch is An amino acid sequence is provided which is as selected from one of c), d), e) and f).

Even more preferably, the amino acids of the invention comprise three or more amino acid residue stretches, wherein the first amino acid residue stretch comprises
a) SEQ ID NO: 98,
b) An amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 98, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (such as measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference an amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Consisting of stretches of amino acid residues, which should have about the same or higher potency (as defined herein) It is selected from,
The second amino acid residue stretch is
c) SEQ ID NO: 102,
d) a stretch of amino acid residues having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 102,
i) the amino acid residue stretch has aspartic acid (Asp, D) at position 6 (position 54 determined by Kabat numbering),
ii) the amino acid sequence comprising the amino acid residue stretch has the same, substantially the same or higher affinity as compared to the amino acid sequence comprising the amino acid residue stretch having no three, two or one amino acid difference ( The affinity is as measured by surface plasmon resonance) and binds to protein F of hRSV and / or the amino acid sequence comprising the amino acid residue stretch has three, two or one amino acid differences. Selected from the group consisting of amino acid residue stretches that have the same, approximately the same, or higher potency (as defined herein) as compared to an amino acid sequence comprising the above amino acid residue stretches without And
The third amino acid residue stretch is
e) an amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 121, said amino acid sequence comprising said amino acid residue stretch Has the same, nearly the same, or a higher affinity compared to an amino acid sequence comprising the amino acid residue stretch without three, two or one amino acid difference (the affinity is measured by surface plasmon resonance An amino acid sequence that binds to protein F of hRSV and / or contains an amino acid residue stretch that does not have three, two or one amino acid difference Amino acids that have the same, about the same, or higher potency (as defined herein) as compared to the sequence Noic acid residue stretch.

The specific amino acid residues stretch comprising one or more amino acid sequences of, for example, (as further described herein (actual or apparent) K _D values (actual or apparent) K _A values, k _on rate and / or _{k off} rate, or alternatively is preferably measured as _{IC 50} values, and / or represented by) improved binding characteristics, improved affinity for a protein F of hRSV and / or avidity And / or improved properties such as improved efficacy and / or efficacy of neutralizing hRSV.

More specifically, the amino acid sequence of the present invention containing one or more specific amino acid residue stretches described above is preferably 1000 nM to 1 nM or less, preferably 100 nM to 1 nM or less, more preferably 15 nM to 1 nM or more. More preferably binds to protein F of hRSV with a dissociation constant (K _D ) of 10 nM to 1 nM or less and / or from 10 ⁴ M ⁻¹ s ⁻¹ to about 10 ⁷ M ⁻¹ s ⁻¹ , preferably 10 ^{5 M -1 s -1 ~10 7 M} -1 s -1, more preferably such as to bind the protein F of hRSV at about ¹⁰ 6 ^M -1 ^{s -1} or more _{k on} rate, and / or 10 ^{- 2} s ^-1 (t _1/2 = 0.69 s) to 10 ^-4 s ^-1 (in the case of a substantially irreversible complex in which t _1/2 is several days), preferably 10 ^-3 s ^-1 10 ⁴ s ^-1 as binding to protein F of hRSV in the following _{k off} rate,
(As further described herein (actual or apparent) _{K D} value, (actual or apparent) _{K A value k on} rate and / or _{k off} rate, or alternatively suitable as an _{IC 50} value Can bind to protein F of hRSV with an affinity (measured and / or expressed).

  Some preferred IC50 values for the binding of the amino acid sequence of the invention to protein F of hRSV will become clear from the further description and examples herein.

  Assays for determining the IC50 include competition assays such as competition ELISAs (eg, competition with Synagis® or Fab fragments thereof), or more preferably Anderson et al. (1985, J. Clin. Microbiol. 22: 1050). -1052, 1988, J. Virol. 62: 4232-4238), a neutralization assay such as a modification of this assay as described in Example 6, or for example, Johnson et al. (1997, J. Inf. Dis. 176: 1215-1224), and variations thereof.

  For example, in a competition assay with a Fab fragment of Synagis®, an amino acid sequence of the invention may have an IC50 value of 1 nM to 100 nM, preferably 10 nM to 50 nM or less.

  For example, in a microneutralization assay against hRSV Long (eg, as described in Example 6), an amino acid sequence of the invention may have an IC50 value of 100 nM to 1000 nM, preferably 100 nM to 500 nM or less.

  The present invention also relates to the origin of the amino acid sequence of the invention (or the nucleotide sequence of the invention used to express it) and to produce or obtain (or to produce or obtain the amino acid sequence or nucleotide sequence of the invention) It should be noted that the method is not limited. Thus, the amino acid sequences of the invention can be natural amino acid sequences (from any suitable species) or synthetic or semi-synthetic amino acid sequences.

  In the broadest sense, these stretches of amino acid residues (as defined herein) may cause the amino acid sequence of the present invention to have a certain affinity and / or specificity for antigenic site II on protein F of hRSV. It should be noted that these amino acid residue stretches are not limited to the particular structural role or function that they may have in the amino acid sequences of the present invention, as long as they can be attached in an efficient manner. Thus, in general, the present invention provides in the broadest sense that the entire amino acid sequence forms a binding domain and / or binding unit capable of binding to antigenic site II on protein F of hRSV. One or more amino acid residue stretches as defined herein (especially 2 amino acids) capable of binding antigen site II and suitably linked to one another via one or more additional amino acid sequences. Suitable combinations of one or more such stretches of amino acid residues).

  Such an amino acid sequence can be, for example, a suitable "protein scaffold" comprising at least one amino acid residue stretch as defined herein (ie, as part of its antigen binding site). Suitable scaffolds for displaying amino acid sequences will be apparent to those of skill in the art, such as, but not limited to, binding scaffolds based on or derived from immunoglobulins, protein scaffolds from the Protein A domain (eg, Affibodies ) (TM)), tendamistat, fibronectin, lipocalin, CTLA-4, T cell receptor, designed ankyrin repeats, avimers and PDZ domains (Binz et al., 2005, Nat. Biotech. 23: 1257). And binding moieties based on DNA or RNA, including but not limited to DNA or RNA aptamers (Ulrich et al., 2006, Comb. Chem. High Throughput Screen 9 (8): 619-32). It is.

Also, any amino acid sequence of the invention comprising one or more amino sequence stretches as defined herein with respect to the amino acid sequence of the invention may be associated with hRSV protein F (as defined herein). ) such that it can specifically bind, and more specifically is as defined herein (as further described herein (actual or apparent) K _D value (actual or apparent) _{K a value k on} rate and / or _{k off} rate, or is suitably measured as an alternative to _{an IC 50} value, and / or represented by) a protein F of hRSV affinity Preferably, they are capable of binding. Any amino acid sequence of the invention comprising one or more amino acid residue stretches as defined herein with respect to the amino acid sequence of the invention may be as defined herein (as defined herein). Preferably, it is one that is capable of neutralizing hRSV with potency (as measured in any suitable assay).

  Furthermore, one or more of the CDRs as defined herein with respect to the amino acid sequences of the invention may be "grafted" onto another "scaffold", including but not limited to a human scaffold or a non-immunoglobulin scaffold. It will be apparent to those skilled in the art that it may be possible to do so. Suitable scaffolds and techniques for such CDR grafting will be apparent to those of skill in the art and are known in the art. For example, US Pat. No. 7,180,370, WO 01/27160, EP 0605522, EP 0460167, US Pat. No. 7,054,297, Nicaise et al., Protein Science. 2004), 13: 1882-1891, Ewert et al., Methods, 2004 Oct; 34 (2): 184-199), Kettleborough et al., Protein Eng. 1991 Oct; 4 (7): 773-783, O ' Brienand Jones, Methods Mol. Biol. 2003: 207: 81-100, and Skerra, J. Mol. Recognit. 2000: 13: 167-187, and Saerens et al., J. Mol. Biol. 2005 Sep 23; 352. See (3): 597-607, and further references cited therein. For example, techniques known per se for grafting mouse or rat CDRs onto human frameworks and scaffolds include one or more of the CDR sequences as defined herein with respect to the amino acid sequences of the invention, plus one Alternatively, it can be used to produce chimeric proteins comprising multiple human framework regions or sequences.

In one specific, but non-limiting embodiment, the amino acid sequence of the invention comprises an amino acid sequence comprising an immunoglobulin fold or an immunoglobulin fold under suitable (eg, physiological) conditions (ie, by folding). Amino acid sequence. See especially the review by Halaby et al. (1999, J. Protein Eng. 12: 563-71). Preferably, when properly folded to form an immunoglobulin fold, the stretch of amino acid residues can properly form an antigen binding site for binding to specific antigen site II on protein F of hRSV. , More preferably as defined herein (K _D values (actual or apparent), K _A values (actual or apparent), kon rates and k _on rates as further described herein) And / or k _off rate, or alternatively, is suitably measured and / or expressed as an IC ₅₀ value) and can bind antigen site II on protein F of hRSV.

  In another specific, but non-limiting embodiment, the amino acid sequence of the invention is an immunoglobulin sequence. Specifically, but not exclusively, the amino acid sequences of the invention consist essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively). Or any suitable fragment of such amino acid sequence that further binds to antigen site II on protein F of hRSV.

  In such amino acid sequences of the invention, the framework sequence may be any suitable framework sequence, examples of suitable framework sequences are, for example, mentioned in standard handbooks, as well as herein. It will be apparent to those skilled in the art based on further disclosure and the prior art.

The framework sequence is preferably an immunoglobulin framework sequence, or a suitable combination of framework sequences derived (eg, by humanization or camelization) from an immunoglobulin framework sequence. For example, the framework sequence can be a framework sequence derived from a light chain variable domain (eg, a _VL sequence) and / or a heavy chain variable domain (eg, a _VH sequence). When the amino acid sequence of the present invention is a heavy chain variable domain sequence, the heavy chain variable domain sequence derived from a conventional four-chain antibody (for example, but not limited to, a _VH sequence derived from a human antibody) or a so-called (herein referred to as a _VH sequence). It may be a so-called V _HH sequence (as defined herein) from a "defined" heavy chain antibody. In one particularly preferred aspect, the framework sequence is derived from a V _HH sequence (the framework sequence can optionally be partially or fully humanized) or camelized (as defined herein). Any framework sequence that is a conventional _VH sequence.

  For a review of heavy chain antibodies and their variable domains, see in particular the prior art cited herein, and the prior art mentioned on page 59 of WO 08/020079, and WO 06 / WO of the international application. Reference is made to the list of references cited on pages 41 to 43 of U.S. Pat. No. 4,015,153 (prior art and references are incorporated herein by reference).

The framework sequence is that the amino acid sequence of the present invention is a domain antibody (or an amino acid sequence suitable for use as a domain antibody), a single domain antibody (or an amino acid sequence suitable for use as a single domain antibody), "dAb (Or amino acid sequences suitable for use as dAbs), or Nanobodies® (including but not limited to _VHH sequences). Also, suitable framework sequences will be apparent to those skilled in the art, for example, based on standard handbooks, as well as the additional disclosure and prior art referred to herein.

In particular, the framework sequences present in the amino acid sequences of the present invention may be modified such that the amino acid sequence of the present invention is Nanobody® (WO 08/020079 (Tables A-3 to A-8)). (As defined above). Some preferred but non-limiting examples of (suitable combinations of) such framework sequences will become clear from the further disclosure herein (see, for example, Table A-6). Generally Nanobodies (in particular _{V HH} sequences and (partially) humanized Nanobodies (TM)) is particularly (e.g. further described in the third line 61, page 24 line 98, page WO 08/020079 May be characterized by the presence of one or more "characteristic residues" in one or more framework sequences.

  As already described herein, the amino acid sequence and structure of Nanobody® can be considered to be composed of, but not limited to, four framework regions or “FRs” Represent "framework region 1" or "FR1", "framework region 2" or "FR2", "framework region 3" or "FR3" and "framework region 4" in the art and herein, respectively. Or "FR4", between which three complementarity determining regions or "CDRs" are inserted, which in the art are referred to in the art as "complementarity determining region 1" or "CDR1", respectively. "," Complementarity determining region 2 "or" CDR2 "and" Complementarity determining region 3 "or" CDR3 ". Some preferred framework sequences and CDRs (and combinations thereof) present in the Nanobodies® of the invention are as described herein.

For this reason, Nanobodies® generally have the (general) structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
(Where FR1 to FR4 represent framework regions 1 to 4 respectively, and CDR1 to CDR3 represent complementarity determining regions 1 to 3 respectively, and one or more characteristic residues Is as defined further herein)).

In this regard, the amino acid sequences of the present invention may consist essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively). CDR2 is
a) SEQ ID NO: 102,
b) a stretch of amino acid residues having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 102,
i) the amino acid residue stretch has aspartic acid (Asp, D) at position 6 (position 54 determined by Kabat numbering); ii) three or two amino acid sequences containing the amino acid residue stretch Or with the same, about the same, or a higher affinity (such as measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference an amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Choose from stretches of amino acid residues that should be of about the same or higher potency (as defined herein). Selected.

  These preferred complementarity determining regions (CDR2 sequences) are also referred to as "CDR2 (s) of the invention".

Preferably, the amino acid sequence of the invention may consist essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR2 But,
a) SEQ ID NO: 102,
b) a stretch of amino acid residues having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 102,
i) the amino acid residue stretch has aspartic acid (Asp, D) at position 6 (position 54 determined by Kabat numbering),
ii) the amino acid sequence comprising the amino acid residue stretch has the same, substantially the same or higher affinity as compared to the amino acid sequence comprising the amino acid residue stretch having no three, two or one amino acid difference ( The affinity is as measured by surface plasmon resonance) and binds to protein F of hRSV and / or the amino acid sequence comprising the amino acid residue stretch has three, two or one amino acid differences. Selected from the group consisting of amino acid residue stretches that have the same, approximately the same, or higher potency (as defined herein) as compared to an amino acid sequence comprising the above amino acid residue stretches without Wherein at least one of CDR1 or CDR3 is
c) SEQ ID NO: 98;
d) An amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 98, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (as said affinity is measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference the amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Consisting of stretches of amino acid residues, which should have about the same or higher potency (as defined herein) CDR1, are selected from and / or e) SEQ ID NO: 121,
f) an amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 121, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (such as measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference an amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Stretches of amino acid residues which should be of about the same or higher potency (as defined herein). CDR3 is selected from the group
Is selected from

Even more preferably, the amino acid sequence of the invention may consist essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively); Here CDR1 is
a) SEQ ID NO: 98,
b) An amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 98, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (such as measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference an amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Consisting of stretches of amino acid residues, which should have about the same or higher potency (as defined herein) It is selected from,
CDR2 is
c) SEQ ID NO: 102,
d) a stretch of amino acid residues having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 102,
i) the amino acid residue stretch has aspartic acid (Asp, D) at position 6 (position 54 determined by Kabat numbering),
ii) the amino acid sequence comprising the amino acid residue stretch has the same, substantially the same or higher affinity as compared to the amino acid sequence comprising the amino acid residue stretch having no three, two or one amino acid difference ( The affinity is as measured by surface plasmon resonance) and binds to protein F of hRSV and / or the amino acid sequence comprising the amino acid residue stretch has three, two or one amino acid differences. Selected from the group consisting of amino acid residue stretches that have the same, approximately the same, or higher potency (as defined herein) as compared to an amino acid sequence comprising the above amino acid residue stretches without And
CDR3 is
e) SEQ ID NO: 121,
f) an amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 121, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (such as measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference an amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Stretches of amino acid residues which should be of about the same or higher potency (as defined herein). It is selected from the group.

  In certain aspects, the amino acid sequence or Nanobody® of the invention comprises at least SEQ ID NO: 102.

In another particular aspect, the amino acid sequence or Nanobody® of the invention comprises at least SEQ ID NO: 102,
a) SEQ ID NO: 98,
b) an amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 98, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (as said affinity is measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference the amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; An amino acid residue stretch, which shall have about the same or higher potency (as defined herein);
c) SEQ ID NO: 121,
d) An amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 121, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (as said affinity is measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference the amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Select from stretches of amino acid residues that should have about the same or higher potency (as defined herein). It is the, and at least one amino acid residue stretches (CDR sequence).

  Preferably, the amino acid sequence or Nanobody (R) of the invention is selected from the group consisting of at least SEQ ID NO: 102 and one stretch of amino acid residues as defined in a) and b), and the other stretch is c). And d) at least two amino acid residue stretches (CDR sequences) selected from the group consisting of the defined amino acid residue stretches.

  In another specific aspect, the amino acid sequence or Nanobody® of the invention comprises at least SEQ ID NO: 102 and at least one stretch of amino acid residues (CDR sequence) selected from SEQ ID NO: 98 and SEQ ID NO: 121. including.

  Preferably, the amino acid sequence or Nanobody® of the present invention comprises SEQ ID NO: 98, SEQ ID NO: 102 and SEQ ID NO: 121.

  Also, Table A-6 shows preferred combinations of the CDR1, CDR2, and CDR3 sequences.

  The amino acid sequence of the invention can consist essentially of the heavy chain variable domain sequence from a conventional four chain antibody, or can consist essentially of the heavy chain variable domain sequence from a heavy chain antibody. The amino acid sequence of the present invention may be a domain antibody (or an amino acid sequence suitable for use as a domain antibody), a single domain antibody (or an amino acid sequence suitable for use as a single domain antibody), "dAb" (or dAb Amino acid sequences suitable for use in) or Nanobodies®.

  For a review of (single) domain antibodies, reference is also made to the prior art mentioned above and to EP 0 368 684. With respect to the term "dAb", for example, Ward et al. (1989, Nature 341: 544-6), Holt et al., 2003, Trends Biotechnol, 21: 484-490, and for example, WO 06/030220, Reference is made to WO 06/003388 and other published patent applications of Domantis Ltd. It should also be noted that although not of mammalian origin and less preferred in the context of the present invention, single domain antibodies or single variable domains may be derived from certain species of sharks. (Eg, the so-called “IgNAR domain”, see eg WO 05/18629).

  In particular, the amino acid sequences of the present invention may consist essentially of or may be Nanobodies® (as defined herein) or suitable fragments thereof (Note: Nanobodies®, Nanobodies ( (Registered trademark) and Nanoclone (registered trademark) are registered trademarks of Ablynx NV).

Nanobody® has the (general) structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
(Where FR1 to FR4 represent framework regions 1 to 4 respectively, and CDR1 to CDR3 represent complementarity determining regions 1 to 3 respectively, and one or more characteristic residues Can be defined as an amino acid sequence having as defined in WO 08/020079 (Tables A-3 to A-8).

More specifically, Nanobodies® have the (general) structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
(Where FR1 to FR4 represent framework regions 1 to 4, respectively, and CDR1 to CDR3 represent complementarity-determining regions 1 to 3, respectively);
i) at least one of the amino acid sequences of SEQ ID NO: 60-SEQ ID NO: 76, SEQ ID NO: 138-SEQ ID NO: 141, and SEQ ID NO: 146-SEQ ID NO: 157 (see Table A-4) and at least 80% amino acids Have identity (the amino acid residues forming the CDR sequence are ignored to determine the degree of amino acid identity) (in this regard, SEQ ID NOs: 60-76, 138-141, and The Nanobody® framework 1 sequence (SEQ ID NOs: 81-97 and SEQ ID NO: 166) and the framework 2 sequence (SEQ ID NO: 99) of SEQ ID NOs: 146-157 (see Table A-4) To SEQ ID NO: 100), the framework 3 sequence (SEQ ID NO: 103 to SEQ ID NO: 120 and SEQ ID NO: 167 to SEQ ID NO: 168), and Muwaku 4 sequences also see Table A-6, which (SEQ ID NO: 123 and SEQ ID NO: 169) are listed),
ii) Preferably one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to Kabat numbering are , An amino acid sequence selected from the characteristic residues mentioned in Tables A-3 to A-8 of WO 08/020079.

  For a further review of Nanobodies®, reference is made to the prior art mentioned in the present invention, for example as described in WO 08/020079 (p. 16).

Such Nanobodies® can be derived in any suitable manner from any suitable source, such as natural (ie, derived from a species of camelid) V _HH sequence or synthetic or It may be a semi-synthetic amino acid sequence.

Also, such Nanobodies® can be derived from any suitable source in any suitable manner, such as a naturally occurring (ie, from a suitable species of camelid) V _HH sequence, or It may be a synthetic or semi-synthetic amino acid sequence, including but not limited to “humanized” Nanobodies® (as defined herein), “camel” (as defined herein). "Immunized" immunoglobulin sequences (particularly camelized heavy chain variable domain sequences), as well as affinity maturation (eg, starting from synthetic, random or natural immunoglobulin sequences), CDR grafting, as further described herein. , Veneering, ligation of fragments from various immunoglobulin sequences, PCR assembly using overlapping primers, and immunoglobulin sequences known to those of skill in the art. Nanobodies® obtained by similar techniques to manipulate, or techniques such as any suitable combination of any of the above are included. Also, where the Nanobodies® include a _VHH sequence, the Nanobodies® may provide one or more additional (partially or fully) humanized Nanobodies® of the present invention. , Can be suitably humanized as further described herein. Similarly, where a Nanobody® comprises a synthetic or semi-synthetic sequence (eg, a partially humanized sequence), the Nanobody® again may comprise one or more additional (part or Optionally, it can also be more suitably humanized as described herein to provide a (fully) humanized Nanobody®.

In particular, the humanized Nanobody (R) can be generally an amino acid sequence as defined for Nanobody (R) in the preceding paragraphs, but wherein the humanized substitution (as defined herein) is included. And / or at least one amino acid residue corresponding to a humanized substitution is present (especially at least one of the framework residues). Based on the disclosure herein, some preferred but non-limiting humanized substitutions (and suitable combinations thereof) will be apparent to those skilled in the art. Additionally or alternatively, by comparing the sequence of the framework region of the native V _HH sequence to the corresponding framework sequence of one or more closely related human V _H sequences, Useful humanized substitutions can be identified, and one or more of the potentially useful humanized substitutions (or combinations thereof) determined in this manner are then _added to the _VHH sequence (as known per se). And the resulting humanized _VHH sequence can be introduced into a target according to any of the methods described above, wherein the affinity, stability, ease and level of expression, and / or It can be tested for other desired properties. Thus, by suitable trial and error, other suitable humanized substitutions (or suitable combinations thereof) can be determined by those skilled in the art based on the disclosure herein. Further, based on the above, Nanobody (registered trademark) (the framework region thereof) can be partially humanized or fully humanized.

In this regard, some preferred Nanobodies® of the invention specifically bind (as further defined herein) to protein F of hRSV,
i) a humanized variant of the amino acid sequence having SEQ ID NO: 5 (see Table A-1) and / or ii) of the amino acid sequence of SEQ ID NO: 5 (see Table A-1) And / or at least one of the amino acid sequences of SEQ ID NOs: 60-76, 138-141, and 146-157 (see Table A-4). Has at least 80% amino acid identity (ignoring the amino acid residues forming the CDR sequence to determine the degree of amino acid identity);
iii) Preferably one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to Kabat numbering are international Selected from the characteristic residues referred to in Tables A-3 to A-8 of Publication 08/020079.
Nanobody (registered trademark).

  The present invention provides many humanized and / or sequence-optimized amino acid sequences and / or Nanobodies® that are particularly suitable for binding to protein F of hRSV.

Therefore, in one embodiment of the present invention,
a) SEQ ID NO: 60 to SEQ ID NO: 76
b) an amino acid sequence having only one amino acid difference from one of SEQ ID NO: 60 to SEQ ID NO: 76, preferably only two, more preferably only one;
i) the amino acid sequence has glutamine (Gln, Q) at position 105 (the position is determined by Kabat numbering);
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) (As defined herein), and / or an Nanobody® selected from the amino acid sequences.

  In a preferred aspect, the amino acid sequence and / or Nanobody® of the invention comprises or consists essentially of one of SEQ ID NO: 60-SEQ ID NO: 76.

In another aspect, the invention provides:
a) SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76
b) only three, preferably only two and more preferably only one amino acid difference from one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75 and SEQ ID NO: 76 An amino acid sequence having
i) the amino acid sequence has glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and / or arginine (Arg, R) at position 83 (the above positions are determined by Kabat numbering; Desired),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) (As defined herein), and / or Nanobodies® selected from amino acid sequences.

In a preferred embodiment, the amino acid sequence and / or Nanobody® comprises
a) SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76
b) only three, preferably only two and more preferably only one amino acid difference from one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75 and SEQ ID NO: 76 An amino acid sequence having
i) the amino acid sequence has glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and arginine (Arg, R) at position 83 (the above positions are determined by Kabat numbering) ),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) (As defined herein).

  Preferred amino acid sequences and / or Nanobodies® of the invention comprise one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75 and SEQ ID NO: 76, or From this it becomes essentially.

In yet another aspect, the invention provides a method comprising:
a) SEQ ID NO: 65 and SEQ ID NO: 76,
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NO: 65 and SEQ ID NO: 76,
i) the amino acid sequence is aspartic acid (Asp, D) at position 54, glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and / or arginine (Arg, R) at position 83; (The positions are determined by Kabat numbering); ii) the amino acid sequence has the same, nearly the same, or a higher affinity compared to an amino acid sequence that does not have three, two or one amino acid differences. An amino acid that binds to protein F of hRSV and / or whose amino acid sequence does not have three, two or one amino acid difference, wherein the affinity is as measured by surface plasmon resonance An amino acid sequence selected from an amino acid sequence and / or having the same, about the same, or higher potency (as defined herein) as compared to the sequence; Alternatively, Nanobody (registered trademark) is provided.

In a preferred embodiment, the amino acid sequence and / or Nanobody® comprises
a) SEQ ID NO: 65 and SEQ ID NO: 76,
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NO: 65 and SEQ ID NO: 76,
i) the amino acid sequence has aspartic acid (Asp, D) at position 54, glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and arginine (Arg, R) at position 83; (The above position is obtained by Kabat numbering)
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) (As defined herein).

  Preferably, an amino acid sequence and / or Nanobody® of the invention comprises or consists essentially of SEQ ID NO: 65. In another preferred embodiment, the amino acid sequence and / or Nanobody® of the invention comprises or consists essentially of SEQ ID NO: 76. In another preferred embodiment, the amino acid sequence and / or Nanobody® of the invention comprises or consists essentially of SEQ ID NO: 76.

The present invention
a) SEQ ID NO: 146 to SEQ ID NO: 153
b) an amino acid sequence having only one, preferably two, more preferably only one amino acid difference from one of SEQ ID NOs: 146 to 153,
i) the amino acid sequence comprises proline (Pro, P) at position 14, arginine (Arg, R) at position 19, leucine (Leu, L) at position 20 and / or leucine (Leu, L) at position 108 Have (the above positions are determined by Kabat numbering),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) Also provided are a number of humanized and / or sequence-optimized amino acid sequences and / or Nanobodies® selected from amino acid sequences (as defined herein).

  In a preferred embodiment, the amino acid sequence and / or Nanobody® of the invention comprises or consists essentially of one of SEQ ID NO: 146-SEQ ID NO: 153.

In another aspect, the invention provides:
a) SEQ ID NO: 146 to SEQ ID NO: 149 and SEQ ID NO: 151 to SEQ ID NO: 153, b) One of SEQ ID NO: 146 to SEQ ID NO: 149 and SEQ ID NO: 151 to SEQ ID NO: 153 and only three, preferably only two, More preferably an amino acid sequence having only one amino acid difference,
i) the amino acid sequence comprises proline (Pro, P) at position 14, arginine (Arg, R) at position 19, leucine (Leu, L) at position 20, and / or leucine (Leu, L) at position 108 Having arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85, and / or glutamine (Gln, Q) at position 105;
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) (As defined herein), and / or Nanobodies® selected from amino acid sequences.

In a preferred embodiment, the amino acid sequence and / or Nanobody® comprises
a) SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153,
i) the amino acid sequence is
SEQ ID NO: 146 (this amino acid sequence preferably has glutamine (Gln, Q) at position 105);
SEQ ID NO: 147 (this amino acid sequence preferably has arginine (Arg, R) at position 83 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 148 (this amino acid sequence preferably has glutamic acid (Glu, E) at position 85 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 149 (this amino acid sequence preferably has arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 151 (this amino acid sequence preferably has arginine (Arg, R) at position 83);
SEQ ID NO: 152 (this amino acid sequence preferably has glutamic acid (Glu, E) at position 85);
SEQ ID NO: 153 (this amino acid sequence preferably has arginine (Arg, R) at position 83 and glutamic acid (Glu, E) at position 85)
The amino acid sequence has a proline (Pro, P) at position 14 and an arginine (Arg, R) at position 19, as in the case of having only 3, preferably 2, and more preferably only 1 amino acid difference. , Leucine (Leu, L) at position 20 and / or leucine (Leu, L) at position 108, arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85, and / or 105. Glutamine (Gln, Q) at the position (the position is determined by Kabat numbering),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) (As defined herein).

  Preferred amino acid sequences and / or Nanobodies® of the invention comprise or consist essentially of one of SEQ ID NOs: 146-149 and SEQ ID NOs: 151-153.

The amino acid sequences and / or Nanobodies® of the invention exhibit reduced immunogenicity upon administration to a human subject. In addition, the amino acid sequences and / or Nanobodies® of the present invention can be used to compare the corresponding parent amino acid sequence to the protein F of hRSV (further (actual or apparent) as described herein (actual or apparent)). shows _D value, the (actual or apparent) _{K a value k on} rate and / or _{k off} rate, or alternatively is preferably measured as _{IC 50} values, and / or represented by) improved binding properties (International application PCT / EP2009 / 056975 filed by Ablynx NV on June 5, 2009 entitled "Amino acid sequences directed against envelope proteins of avirus and polypeptides comprising the same for the treatment of viraldiseases" like).

  In making the Nanobodies of the present invention, high levels of pyroglutamic acid (pGlu) at the amino terminus were detected by RP-HPLC. After the fermentation, more than 15% of the pGlu level was detected, and during the stability test the pGlu level increased steadily during storage. Such changes cause inhomogeneities in the final product that need to be avoided. Thus, controlling / blocking pGlu formation is important in maintaining therapeutic proteins within set specifications. Certain liquid formulations and / or storage conditions that minimize the formation of pyroglutamic acid are required for proteins with N-terminal glutamate.

  In the present invention, the possibility of N-terminal pGlu post-translational modification by changing N-terminal glutamic acid (E) [HOOC- (CH2) 2-protein] to aspartic acid (D) [HOOC-CH2-protein] Was eliminated and the stability of the product increased. Accordingly, the present invention also relates to such amino acid sequences and Nanobodies wherein the glutamic acid at position 1 (the position is determined by Kabat numbering) has been changed to aspartic acid.

The present invention provides a number of sequence-optimized amino acid sequences and / or Nanobodies® that exhibit increased stability upon storage during stability testing. Therefore, in one embodiment of the present invention,
a) SEQ ID NO: 138 to SEQ ID NO: 141 and SEQ ID NO: 154 to SEQ ID NO: 157;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 138 to 141 and 154 to 157,
i) the amino acid sequence has aspartic acid (Asp, D) at position 1 (the position is determined by Kabat numbering);
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) (As defined herein), and / or an Nanobody® selected from the amino acid sequences.

  In a preferred embodiment, an amino acid sequence and / or Nanobody® of the invention comprises or consists essentially of one of SEQ ID NOs: 138-141 and SEQ ID NOs: 154-157.

  In another aspect, the amino acid sequences and / or Nanobodies® of the invention comprise or consist essentially of SEQ ID NO: 5, wherein Glu at position 1 has been changed to Asp.

  In another aspect, the amino acid sequence and / or Nanobody® of the invention comprises or consists essentially of SEQ ID NO: 62 wherein Glu at position 1 has been changed to Asp.

  In another aspect, the amino acid sequences and / or Nanobodies® of the invention comprise or consist essentially of SEQ ID NO: 65 wherein Glu at position 1 has been changed to Asp.

  In another aspect, the amino acid sequence and / or Nanobody® of the invention comprises or consists essentially of SEQ ID NO: 76, wherein Glu at position 1 has been changed to Asp.

  In another aspect, the amino acid sequence and / or Nanobody® of the invention comprises, or consists essentially of, one of SEQ ID NOs: 146-153 wherein Glu at position 1 has been changed to Asp. Become

  Preferably, an amino acid sequence and / or Nanobody® of the invention comprises or consists essentially of SEQ ID NO: 138. In another preferred aspect, the amino acid sequence and / or Nanobody® of the invention comprises or consists essentially of SEQ ID NO: 139. In another preferred embodiment, the amino acid sequence and / or Nanobody® of the invention comprises or consists essentially of SEQ ID NO: 140. In another preferred aspect, the amino acid sequence and / or Nanobody® of the invention comprises or consists essentially of SEQ ID NO: 141. In another preferred aspect, the amino acid sequence and / or Nanobody® of the invention comprises or consists essentially of one of SEQ ID NOs: 154-157.

  In another aspect, the amino acid sequence and / or Nanobody® of the invention comprises one or more (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) amino acid residues are mutated from Val5Leu, Ala14Pro, Ser19Arg, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and A1 selected from Gly108Leu. Includes or consists essentially of SEQ ID NO: 5.

  In another aspect, the amino acid sequence and / or Nanobody® of the present invention comprises one or more (eg, 2, 3, 4, 5, 6, 7, 8, or 9) Comprises or consists essentially of SEQ ID NO: 5 with a mutation selected from Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp.

  In another aspect, the amino acid sequences and / or Nanobodies® of the invention have one or more (eg, two, three or four) amino acid residues from Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu. Includes or consists essentially of SEQ ID NO: 5, with the selected mutation.

  In another aspect, the amino acid sequences and / or Nanobodies® of the invention have one or more (eg, 2, 3, 4, 5, 6, or 7) amino acid residues. Includes or consists essentially of SEQ ID NO: 5, mutated from Ala14Pro, Ser19Arg, Ile20Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu.

  In another aspect, the amino acid sequence and / or Nanobody® of the invention comprises one or more (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) amino acid residues are mutated from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and GlyA from Gly54A. Comprises or consists essentially of SEQ ID NO: 5, wherein Glu at position 1 has been changed to Asp.

  In another aspect, the amino acid sequence and / or Nanobody® of the present invention comprises one or more (eg, 2, 3, 4, 5, 6, 7, 8, or 9) Has undergone a mutation selected from Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp, and Glu at position 1 has been changed to Asp. Or consists essentially of.

  In another aspect, the amino acid sequences and / or Nanobodies® of the invention have one or more (eg, two, three or four) amino acid residues from Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu. Includes or consists essentially of SEQ ID NO: 5, which has undergone a selected mutation and wherein Glu at position 1 has been changed to Asp.

  In another aspect, the amino acid sequences and / or Nanobodies® of the invention have one or more (eg, 2, 3, 4, 5, 6, or 7) amino acid residues. It has a mutation selected from Ala14Pro, Ser19Arg, Ile20Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu, wherein Glu at position 1 has been changed to Asp, comprising or consisting essentially of SEQ ID NO: 5. .

Preferably, the amino acid sequence and / or Nanobody (registered trademark) of the present invention has an amino acid residue
Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln and Gln108Leu,
Ala83Arg, Asp85Glu, Arg105Gln and Gln108Leu,
Gly78Leu, Ala83Arg, Asp85Glu and Arg105Gln,
Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu and Gly54Asp,
Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu and Gly54Asp,
Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln and Gly54Asp,
Gly54Asp,
Ala14Pro, Ser19Arg, Ile20Leu and Gln108Leu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu and Ala83Arg,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu and Asp85Glu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu and Arg105Gln,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg and Asp85Glu, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg and Arg105Gln,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Asp85Glu and Arg105Gln,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, Asp85Glu and Arg105Gln,
Glu1Asp,
Glu1Asp, Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln and Gln108Leu,
Glu1Asp, Ala83Arg, Asp85Glu, Arg105Gln and Gln108Leu,
Glu1Asp, Gly78Leu, Ala83Arg, Asp85Glu and Arg105Gln,
Glu1Asp, Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu and Gly54Asp,
Glu1Asp, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu and Gly54Asp,
Glu1Asp, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln and Gly54Asp,
Glu1Asp and Gly54Asp,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu and Gln108Leu,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu and Ala83Arg,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu and Asp85Glu,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu and Arg105Gln,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg and Asp85Glu,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg and Arg105Gln,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Asp85Glu and Arg105Gln, or Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, AsAg85Gu, AsAg85Ag
Comprising or consisting essentially of SEQ ID NO: 5, mutated to

The amino acid sequences and / or Nanobodies® of the present invention can have, for example, improved stability, reduced immunogenicity, compared to the corresponding wild-type amino acid sequence (as further described herein (see actual or apparent) _{K D} value, (actual or apparent) _{K a value k on} rate and / or _{k off} rate, or is suitably measured as an alternative to _{an IC 50} value, and / or represented) Exhibits improved properties such as improved binding properties, improved affinity of hRSV for protein F and / or improved binding activity, and / or improved efficacy and / or potency to neutralize hRSV ("Amino acid sequences PCT / EP2009 / 056 filed by Ablynx NV on June 5, 2009 entitled "Directed against envelope proteins of avirus and polypeptides comprising the same for the treatment of viraldiseases" As described in No. 75).

More specifically, the amino acid sequence and / or Nanobody (registered trademark) of the present invention is preferably 1000 nM to 1 nM, preferably 100 nM to 1 nM, more preferably 15 nM to 1 nM, or even more preferably 10 nM to 1 nM. It binds to protein F of hRSV with the following dissociation constant (K _D ) and / or from 10 ⁴ M ⁻¹ s ⁻¹ to about 10 ⁷ M ⁻¹ s ⁻¹ , preferably 10 ⁵ M ⁻¹ s ^{−. 1 ~10 7} M ^-1 s ^-1, more preferably such as to bind the protein F of hRSV at about ¹⁰ 6 ^M -1 ^{s -1} or more _{k on} rate, and / or ¹⁰ -2 ^s -1 (t _1/2 = 0.69 s) to 10 ^-4 s ^-1 (in the case of a substantially irreversible complex where t _1/2 is several days), preferably 10 ^-3 s ^-1 to 10 ^-4 s ^-1 the following k such as binding to the protein F of hRSV in _ff rate,
(As further described herein (actual or apparent) _{K D} value, (actual or apparent) _{K A value k on} rate and / or _{k off} rate, or alternatively suitable as an _{IC 50} value Can bind to protein F of hRSV with an affinity (measured and / or expressed).

  Some preferred IC50 values for the binding of the amino acid sequence of the invention to protein F of hRSV will become clear from the further description and examples herein.

  Assays for determining the IC50 include competition assays such as competition ELISAs (eg, competition with Synagis® or Fab fragments thereof), or more preferably Anderson et al. (1985, J. Clin. Microbiol. 22: 1050). -1052, 1988, J. Virol. 62: 4232-4238), a neutralization assay such as a modification of this assay as described in Example 6, or for example, Johnson et al. (1997, J. Inf. Dis. 176: 1215-1224), and variations thereof.

  For example, in a competition assay with a Fab fragment of Synagis®, an amino acid sequence of the invention may have an IC50 value of 1 nM to 100 nM, preferably 10 nM to 50 nM or less.

  For example, in a microneutralization assay against hRSV Long (eg, as described in Example 6), an amino acid sequence of the invention may have an IC50 value of 100 nM to 1000 nM, preferably 100 nM to 500 nM or less.

  The amino acid sequences and Nanobodies® provided by the invention may be in essentially isolated form (as defined herein) or may comprise one or more amino acid sequences or Nanobodies of the invention (as defined herein). (Registered trademark), or may consist essentially of it, and may optionally further comprise one or more additional amino acid sequences or Nanobodies (all optionally one or more). Forming a part of a polypeptide of the invention (also referred to as "polypeptide of the invention" or "construct of the invention", both used interchangeably), linked via a suitable linker of preferable.

  Thus, in another aspect, the invention provides a polypeptide comprising or consisting essentially of one or more of the amino acid sequences or Nanobodies® (or suitable fragments thereof) of the invention (as defined herein). (Also referred to as "the polypeptide of the present invention").

  The method of designing / selecting and / or preparing the polypeptide of the present invention from the amino acid sequence or Nanobody (registered trademark) of the present invention "formats" the amino acid sequence or Nanobody (registered trademark) of the present invention described above in the present specification. The amino acid sequence or Nanobody (registered trademark) of the present invention, which is also referred to as "formatting" and forms a part of the polypeptide of the present invention, is "formatted" or the polypeptide of the present invention. In the format of ". Examples of methods by which the amino acid sequences or Nanobodies® of the present invention can be formatted and examples of such formats will be apparent to those skilled in the art based on the disclosure herein, and such formatted The amino acid sequence or Nanobody® forms a further aspect of the invention.

  For example, but not limited to, one or more of the amino acid sequences or Nanobodies® of the present invention may be monovalent, multivalent, multiparatopic or multispecific according to the invention as described herein. May include one or more additional amino acid sequences that can optionally serve as a binding unit (ie, against the same or another epitope on protein F of hRSV and / or (For one or more antigens, proteins or targets other than protein F) of such polypeptides. Thus, the present invention also relates to a polypeptide which is a monovalent polypeptide or construct comprising or consisting essentially of the amino acid sequence or Nanobody® of the present invention. Thus, the present invention also relates to polypeptides which are multivalent polypeptides or constructs, such as divalent or trivalent polypeptides or constructs. The invention also relates to polypeptides such as multispecific polypeptides or constructs, such as bispecific or trispecific polypeptides or constructs. The invention also relates to polypeptides such as multiparatopic polypeptides or constructs, such as double or triple paratopic polypeptides or constructs.

  Thus, according to a preferred but non-limiting embodiment, an amino acid sequence or Nanobody® of the invention comprises at least one additional amino acid sequence or Nanobody®, whereby at least two, for example, two, Resulting in a polypeptide of the invention comprising one, three, four, five or more amino acid sequences or Nanobodies®, wherein said amino acid sequence or Nanobodies® is optionally (as used herein) (As defined) the linkage may be via one or more linker sequences. A polypeptide of the present invention comprising two or more amino acid sequences or Nanobodies®, wherein at least one, and preferably all are the amino acid sequences or Nanobodies® of the present invention, is referred to herein as a polypeptide of the present invention. And the amino acid sequence or Nanobody® present in such polypeptides is also referred to herein as a “multivalent format”. For example, a “divalent” polypeptide of the invention comprises two amino acid sequences and / or Nanobodies® optionally linked via a linker sequence, and a “trivalent” polypeptide of the invention optionally comprises Including three amino acid sequences and / or Nanobodies® linked via two linker sequences, and so on. Here, at least one of the amino acid sequences and / or Nanobodies (registered trademark) present in the polypeptide, and at most all the amino acid sequences and / or Nanobodies (registered trademark) present in the polypeptide, are the amino acids of the present invention. Sequences and / or Nanobodies®.

  In a multivalent polypeptide of the invention, the two or more amino acid sequences or Nanobodies® may be the same or different and may have the same antigen or antigenic determinant (eg, the same moiety (s) or It may be directed to the epitope (s), or to a different part or epitope, or alternatively to a different antigen or antigenic determinant or any suitable combination thereof. You may. For example, the bivalent polypeptide of the invention may comprise (a) two identical amino acid sequences or a first amino acid sequence directed to a first antigenic determinant of a Nanobody®, a protein or antigen, or Nanobody (registered trademark), and second amino acid sequence or Nanobody (registered trademark) having directivity to the same antigenic determinant of the above protein or antigen different from the first amino acid sequence or Nanobody (registered trademark), (c) protein Alternatively, a first amino acid sequence or Nanobody (registered trademark) having directivity to a first antigenic determinant of an antigen, and a second amino acid sequence or Nanobody (directive) having directivity to another antigenic determinant of the protein or antigen ( ®) or (d) a first amino acid directed to a first protein or antigen. Sequences or Nanobodies (R), and a second may include (i.e., the first differs from the antigen) the second amino acid sequence or Nanobody directed against a protein or antigen (TM). Similarly, trivalent polypeptides of the invention include, but are not limited to, (a) three identical amino acid sequences or Nanobodies®, (b) a first antigenic determinant of an antigen. Two identical amino acid sequences or Nanobodies®, and a third amino acid sequence or Nanobody® having directivity to different antigenic determinants of the same antigen, (c) for the first antigenic determinant of the antigen (D) two identical amino acid sequences or Nanobodies (registered trademark), and a third amino acid sequence or Nanobody (registered trademark) having directivity to a second antigen different from the first antigen, A first amino acid sequence or Nanobody® having directivity to a first antigenic determinant, having directivity to a second antigenic determinant of the first antigen A second amino acid sequence or Nanobody (registered trademark), and a third amino acid sequence or Nanobody (registered trademark) having directivity to a second antigen different from the first antigen, or (e) directing to the first antigen A first amino acid sequence or Nanobody (registered trademark) having a property, a second amino acid sequence or Nanobody (registered trademark) having a directional property to a second antigen different from the first antigen, and the first and It may comprise a third amino acid sequence or a Nanobody® having a tropism for a third antigen different from the second antigen.

  In a preferred embodiment, the invention comprises a polyvalent, preferably bivalent, preferably comprising or consisting essentially of at least two (preferably identical) amino acid sequences or Nanobodies® of the invention (as described above). A multivalent or trivalent polypeptide is provided.

In one aspect, the invention provides:
a) SEQ ID NO: 102,
b) a stretch of amino acid residues having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 102,
i) the amino acid residue stretch has aspartic acid (Asp, D) at position 6 (position 54 determined by Kabat numbering),
ii) the amino acid sequence comprising the amino acid residue stretch has the same, substantially the same or higher affinity as compared to the amino acid sequence comprising the amino acid residue stretch having no three, two or one amino acid difference ( The affinity is as measured by surface plasmon resonance) and binds to protein F of hRSV and / or the amino acid sequence comprising the amino acid residue stretch has three, two or one amino acid differences. An amino acid selected from an amino acid residue stretch that has the same, approximately the same, or a higher potency (as defined herein) as compared to an amino acid sequence comprising the amino acid residue stretch without At least two, preferably identical, amino acids selected from an amino acid sequence containing at least one residue stretch At least three (preferably identical) or comprising the amino acid sequence or Nanobody (TM), or which consists essentially of multivalent, preferably provides a divalent or trivalent polypeptide.

In another aspect, the invention provides that one stretch comprises:
a) SEQ ID NO: 102,
b) a stretch of amino acid residues having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 102,
i) the amino acid residue stretch has aspartic acid (Asp, D) at position 6 (position 54 determined by Kabat numbering),
ii) the amino acid sequence comprising the amino acid residue stretch has the same, substantially the same or higher affinity as compared to the amino acid sequence comprising the amino acid residue stretch having no three, two or one amino acid difference ( The affinity is as measured by surface plasmon resonance) and binds to protein F of hRSV and / or the amino acid sequence comprising said stretch of amino acid residues differs by three, two or one amino acid Is selected from amino acid residue stretches that have the same, approximately the same, or higher potency (as defined herein) as compared to the amino acid sequence comprising the amino acid residue stretch without
At least one stretch,
c) SEQ ID NO: 98;
d) An amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 98, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (as said affinity is measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference the amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; An amino acid residue stretch, which shall have about the same or higher potency (as defined herein);
e) an amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 121, said amino acid sequence comprising said amino acid residue stretch Has the same, nearly the same, or a higher affinity compared to an amino acid sequence comprising the amino acid residue stretch without three, two or one amino acid difference (the affinity is measured by surface plasmon resonance An amino acid sequence that binds to protein F of hRSV and / or contains an amino acid residue stretch that does not have three, two or one amino acid difference Amino acids that have the same, about the same, or higher potency (as defined herein) as compared to the sequence At least two (preferably identical) or at least three (preferably identical) amino acid sequences or Nanobodies selected from amino acid sequences comprising two or more amino acid residue stretches selected from no acid residue stretches (Registered Trademark) or a multivalent, preferably divalent or trivalent polypeptide comprising or consisting essentially of a stretch of amino acid residues corresponding to one of a) and b). As in the amino acid sequence forming part of the multivalent polypeptide, wherein the second stretch of amino acid residues is selected from one of c), d), e) and f). It is something.

Preferred multivalent (eg, divalent or trivalent) polypeptides have a first amino acid residue stretch,
a) SEQ ID NO: 98,
b) An amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 98, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (such as measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference an amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Consisting of stretches of amino acid residues, which should have about the same or higher potency (as defined herein) It is selected from,
The second amino acid residue stretch is
c) SEQ ID NO: 102,
d) an amino acid residue stretch having no more than 3, preferably no more than 2, more preferably only 1 amino acid difference from SEQ ID NO: 102, i) the amino acid residue stretch is at position 6 (determined by Kabat numbering) At position 54) has aspartic acid (Asp, D),
ii) the amino acid sequence comprising the amino acid residue stretch has the same, substantially the same or higher affinity as compared to the amino acid sequence comprising the amino acid residue stretch having no three, two or one amino acid difference ( The affinity is as measured by surface plasmon resonance) and binds to protein F of hRSV and / or the amino acid sequence comprising the amino acid residue stretch has three, two or one amino acid differences. Selected from the group consisting of amino acid residue stretches that have the same, approximately the same, or higher potency (as defined herein) as compared to an amino acid sequence comprising the above amino acid residue stretches without And
The third amino acid residue stretch is
e) SEQ ID NO: 121,
f) an amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 121, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (such as measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference an amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Stretches of amino acid residues which should be of about the same or higher potency (as defined herein). At least two (preferably identical) or at least three (preferably identical) amino acid sequences or Nanobodies® selected from an amino acid sequence comprising a stretch of three or more amino acid residues selected from the group Or may consist essentially of.

In yet another aspect, the invention relates to at least two (preferably, each consisting essentially of four framework regions (FR1 to FR4) and three complementarity determining regions (CDR1 to CDR3, respectively). Polyvalent, preferably divalent or trivalent, polypeptides comprising or consisting essentially of (identical) or at least three (preferably identical) amino acid sequences or Nanobodies®, wherein CDR2 is
a) SEQ ID NO: 102,
b) a stretch of amino acid residues having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 102,
i) the amino acid residue stretch has aspartic acid (Asp, D) at position 6 (position 54 determined by Kabat numbering),
ii) the amino acid sequence comprising the amino acid residue stretch has the same, substantially the same or higher affinity as compared to the amino acid sequence comprising the amino acid residue stretch having no three, two or one amino acid difference ( The affinity is as measured by surface plasmon resonance) and binds to protein F of hRSV and / or the amino acid sequence comprising the amino acid residue stretch has three, two or one amino acid differences. Selected from amino acid residue stretches that have the same, approximately the same, or higher potency (as defined herein) as compared to an amino acid sequence that includes the above amino acid residue stretches having no.

In yet another aspect, the invention relates to at least two (preferably, each consisting essentially of four framework regions (FR1 to FR4) and three complementarity determining regions (CDR1 to CDR3, respectively). Polyvalent, preferably divalent or trivalent polypeptides comprising or consisting essentially of (identical) or at least three (preferably identical) amino acid sequences or Nanobodies®, wherein In CDR2,
a) a stretch of amino acid residues having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 102, or b) SEQ ID NO: 102;
i) the amino acid residue stretch has aspartic acid (Asp, D) at position 6 (position 54 determined by Kabat numbering),
ii) the amino acid sequence comprising the amino acid residue stretch has the same, substantially the same or higher affinity as compared to the amino acid sequence comprising the amino acid residue stretch having no three, two or one amino acid difference ( The affinity is as measured by surface plasmon resonance) and binds to protein F of hRSV and / or the amino acid sequence comprising the amino acid residue stretch has three, two or one amino acid differences. Selected from the group consisting of amino acid residue stretches that have the same, approximately the same, or higher potency (as defined herein) as compared to an amino acid sequence comprising the above amino acid residue stretches without And
At least one of CDR1 or CDR3 is
c) SEQ ID NO: 98;
d) An amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 98, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (as said affinity is measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference the amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Consisting of stretches of amino acid residues, which should have about the same or higher potency (as defined herein) CDR1 selected from,
e) SEQ ID NO: 121, or f) an amino acid residue stretch having only 3, preferably 2, more preferably only 1 amino acid difference from SEQ ID NO: 121, comprising the amino acid residue stretch Has the same, nearly the same, or a higher affinity as compared to an amino acid sequence comprising the amino acid residue stretch without three, two or one amino acid difference (the affinity is measured by surface plasmon resonance An amino acid sequence that binds to protein F of hRSV and / or has an amino acid residue stretch that does not have three, two or one amino acid difference Amino acids that have the same, about the same, or higher potency (as defined herein) as compared to the sequence CDR3 selected from the group consisting of noic acid residue stretches
Is selected from

Preferably, the multivalent (eg, divalent or trivalent) polypeptide consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively) May comprise or consist essentially of at least two (preferably identical) or at least three (preferably identical) amino acid sequences or Nanobodies®,
CDR1 is
a) SEQ ID NO: 98,
b) An amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 98, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (such as measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference an amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Consisting of stretches of amino acid residues, which should have about the same or higher potency (as defined herein) It is selected from, CDR2 is,
c) a stretch of amino acid residues having no more than 3, preferably no more than 2, and more preferably only 1 amino acid difference from SEQ ID NO: 102, or d)
i) the amino acid residue stretch has aspartic acid (Asp, D) at position 6 (position 54 determined by Kabat numbering),
ii) the amino acid sequence comprising the amino acid residue stretch has the same, substantially the same or higher affinity as compared to the amino acid sequence comprising the amino acid residue stretch having no three, two or one amino acid difference ( The affinity is as measured by surface plasmon resonance) and binds to protein F of hRSV and / or the amino acid sequence comprising the amino acid residue stretch has three, two or one amino acid differences. Selected from the group consisting of amino acid residue stretches that have the same, approximately the same, or higher potency (as defined herein) as compared to an amino acid sequence comprising the above amino acid residue stretches without And
CDR3 is
e) SEQ ID NO: 121,
f) an amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 121, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (such as measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference an amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Stretches of amino acid residues which should be of about the same or higher potency (as defined herein). It is selected from the group.

  In certain aspects, the multivalent (eg, divalent or trivalent) polypeptide comprises at least two (preferably identical) or at least three (preferably identical) amino acid sequences or Nanobodies comprising at least SEQ ID NO: 102. (Registered trademark) or may consist essentially of it.

In another specific aspect, the multivalent (eg, divalent or trivalent) polypeptide has at least SEQ ID NO: 102,
c) SEQ ID NO: 98;
d) An amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 98, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (as said affinity is measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference the amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; An amino acid residue stretch, which shall have about the same or higher potency (as defined herein);
e) SEQ ID NO: 121,
f) an amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 121, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (such as measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference an amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Select from stretches of amino acid residues that should have about the same or higher potency (as defined herein). Or at least two (preferably identical) or at least three (preferably identical) amino acid sequences or Nanobodies®, including at least one stretch of amino acid residues (CDR sequences), Or it could be essentially from this.

  Preferred multivalent (eg, divalent or trivalent) polypeptides are selected from the group consisting of at least SEQ ID NO: 102 and one stretch of amino acid residues as defined in c) and d), and the other stretch is e) And f) at least two (preferably identical) or at least three (preferably identical) amino acid residue stretches (CDR sequences) selected from the group consisting of the amino acid residue stretches defined in f). ) Or may consist essentially of or consist of an amino acid sequence or Nanobody®.

  In certain aspects, the multivalent (eg, divalent or trivalent) polypeptide comprises at least two (preferably identical) or at least three (preferably identical), at least SEQ ID NO: 102, SEQ ID NO: 98 and sequence An amino acid sequence or Nanobody (registered trademark) comprising at least one stretch of amino acid residues (CDR sequence) selected from SEQ ID NO: 121, or an amino acid sequence or Nanobody comprising at least SEQ ID NO: 98, SEQ ID NO: 102 and SEQ ID NO: 121 ( (Registered trademark) or may consist essentially of it.

  In another preferred embodiment, the invention comprises a multivalent comprising or consisting essentially of at least three (preferably identical) amino acid sequences or Nanobodies® of the invention (as described above). , Preferably a trivalent polypeptide. In another preferred embodiment, the invention comprises a divalent (consisting essentially of) two (preferably identical) amino acid sequences or Nanobodies of the invention (as described above). A polypeptide is provided. In another preferred embodiment, the present invention comprises a trivalent (consisting essentially of) or consisting essentially of three (preferably identical) amino acid sequences or Nanobodies® of the invention (as described above). A polypeptide is provided.

The present invention
a) SEQ ID NO: 60 to SEQ ID NO: 76
b) an amino acid sequence having only one amino acid difference from one of SEQ ID NO: 60 to SEQ ID NO: 76, preferably only two, more preferably only one;
i) the amino acid sequence has glutamine (Gln, Q) at position 105 (the position is determined by Kabat numbering);
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) Comprising or consisting essentially of at least two (preferably identical) amino acid sequences or Nanobodies® selected from amino acid sequences (as defined herein). Also provided are polyvalent, preferably divalent or trivalent, polypeptides.

  In another preferred aspect, a polypeptide of the invention comprises or consists essentially of at least two identical amino acid sequences or Nanobodies® selected from one of SEQ ID NO: 60-SEQ ID NO: 76. Become

In another preferred aspect, the invention provides a method comprising:
a) SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76
b) only three, preferably only two and more preferably only one amino acid difference from one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75 and SEQ ID NO: 76 An amino acid sequence having
i) the amino acid sequence has glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and / or arginine (Arg, R) at position 83 (the above positions are determined by Kabat numbering; Desired),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) Comprising or consisting essentially of at least two (preferably identical) amino acid sequences or Nanobodies® selected from amino acid sequences (as defined herein). Polyvalent, preferably bivalent or trivalent, polypeptides are provided.

In another preferred aspect, the invention provides a method comprising:
a) SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76
b) only three, preferably only two and more preferably only one amino acid difference from one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75 and SEQ ID NO: 76 An amino acid sequence having
i) the amino acid sequence has glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and arginine (Arg, R) at position 83 (the above positions are determined by Kabat numbering) ),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) Comprising or consisting essentially of at least two (preferably identical) amino acid sequences or Nanobodies® selected from amino acid sequences (as defined herein). Polyvalent, preferably bivalent or trivalent, polypeptides are provided.

  In another preferred aspect, the polypeptide of the present invention comprises at least two of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76. Contain or consist essentially of the same amino acid sequence or Nanobody®.

In another preferred aspect, the invention provides a method comprising:
a) SEQ ID NO: 65 and SEQ ID NO: 76,
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NO: 65 and SEQ ID NO: 76,
i) the amino acid sequence is aspartic acid (Asp, D) at position 54, glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and / or arginine (Arg, R) at position 83; (The positions are determined by Kabat numbering); ii) the amino acid sequence has the same, nearly the same, or a higher affinity compared to an amino acid sequence that does not have three, two or one amino acid differences. An amino acid that binds to protein F of hRSV and / or whose amino acid sequence does not have three, two or one amino acid difference, wherein the affinity is as measured by surface plasmon resonance At least two amino acid sequences selected from amino acid sequences that have the same, approximately the same, or higher potency (as defined herein) as compared to the sequence; Provided are multivalent, preferably divalent or trivalent polypeptides comprising or consisting essentially of (preferably identical) amino acid sequences or Nanobodies®.

In another preferred aspect, the invention provides a method comprising:
a) SEQ ID NO: 65 and SEQ ID NO: 76,
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NO: 65 and SEQ ID NO: 76,
i) the amino acid sequence has aspartic acid (Asp, D) at position 54, glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and arginine (Arg, R) at position 83; (The above position is obtained by Kabat numbering)
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) Comprising or consisting essentially of at least two (preferably identical) amino acid sequences or Nanobodies® selected from amino acid sequences (as defined herein). Polyvalent, preferably bivalent or trivalent, polypeptides are provided.

  In another preferred aspect, the polypeptide of the invention comprises or consists essentially of at least two identical amino acid sequences or Nanobodies® selected from the group consisting of SEQ ID NO: 65 and SEQ ID NO: 76. Become.

The present invention
a) SEQ ID NO: 146 to SEQ ID NO: 153
b) an amino acid sequence having only one, preferably two, more preferably only one amino acid difference from one of SEQ ID NOs: 146 to 153,
i) the amino acid sequence comprises proline (Pro, P) at position 14, arginine (Arg, R) at position 19, leucine (Leu, L) at position 20 and / or leucine (Leu, L) at position 108 Have (the above positions are determined by Kabat numbering),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) Comprising or consisting essentially of at least two (preferably identical) amino acid sequences or Nanobodies® selected from amino acid sequences (as defined herein). Also provided are polyvalent, preferably divalent or trivalent, polypeptides.

  In another preferred aspect, a polypeptide of the invention comprises or consists essentially of at least two identical amino acid sequences or Nanobodies® selected from one of SEQ ID NO: 146-SEQ ID NO: 153. Become

In another preferred aspect, the invention provides a method comprising:
a) SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153,
i) the amino acid sequence comprises proline (Pro, P) at position 14, arginine (Arg, R) at position 19, leucine (Leu, L) at position 20, and / or leucine (Leu, L) at position 108 Having arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85, and / or glutamine (Gln, Q) at position 105;
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) Comprising or consisting essentially of at least two (preferably identical) amino acid sequences or Nanobodies® selected from amino acid sequences (as defined herein). Polyvalent, preferably bivalent or trivalent, polypeptides are provided.

In another preferred aspect, the invention provides a method comprising:
a) SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153,
i) the amino acid sequence is
SEQ ID NO: 146 (this amino acid sequence preferably has glutamine (Gln, Q) at position 105);
SEQ ID NO: 147 (this amino acid sequence preferably has arginine (Arg, R) at position 83 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 148 (this amino acid sequence preferably has glutamic acid (Glu, E) at position 85 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 149 (this amino acid sequence preferably has arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 151 (this amino acid sequence preferably has arginine (Arg, R) at position 83);
SEQ ID NO: 152 (this amino acid sequence preferably has glutamic acid (Glu, E) at position 85);
SEQ ID NO: 153 (this amino acid sequence preferably has arginine (Arg, R) at position 83 and glutamic acid (Glu, E) at position 85)
The amino acid sequence has a proline (Pro, P) at position 14 and an arginine (Arg, R) at position 19, as in the case of having only 3, preferably 2, and more preferably only 1 amino acid difference. Leucine (Leu, L) at position 20 and / or leucine (Leu, L) at position 108, arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85, and / or position 105 Has glutamine (Gln, Q) (the position is determined by Kabat numbering),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) Comprising or consisting essentially of at least two (preferably identical) amino acid sequences or Nanobodies® selected from amino acid sequences (as defined herein). Polyvalent, preferably bivalent or trivalent, polypeptides are provided.

  Preferred polypeptides of the invention comprise, or consist essentially of, at least two identical amino acid sequences or Nanobodies® selected from SEQ ID NOs: 146-149 and SEQ ID NOs: 151-153. Become.

  In another preferred aspect, the invention provides one or more (eg, 2, 3, 4, 5, 6, 7, 8, 9, 9, 10, 11, or 12) Has a mutation selected from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and a mutation selected from the sequence consisting of 5 or any of the following: A multivalent, preferably divalent or trivalent, polypeptide comprising or consisting essentially of at least two (preferably identical) amino acid sequences or Nanobodies®.

  In another preferred embodiment, the present invention provides that one or more (eg, 2, 3, 4, 5, 6, 7, 8, or 9) amino acid residues are Val5Leu, Ala14Pro, At least two (preferably identical) amino acid sequences comprising or consisting essentially of SEQ ID NO: 5, mutated from Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp, or There is provided a multivalent, preferably divalent or trivalent, polypeptide comprising or consisting essentially of Nanobody®.

  In another preferred embodiment, the invention provides a method wherein the one or more (eg, two, three or four) amino acid residues are mutated wherein the amino acid residues are selected from Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu. A polyvalent, preferably divalent or trivalent, comprising or consisting essentially of at least two (preferably identical) amino acid sequences or Nanobodies® comprising or consisting essentially of No. 5 Are provided.

  In another preferred embodiment, the invention provides that one or more (eg, 2, 3, 4, 5, 6, or 7) amino acid residues are Ala14Pro, Ser19Arg, Ile20Leu, Ala83Arg, Asp85Glu, Comprising at least two (preferably identical) amino acid sequences or Nanobodies® comprising or consisting essentially of SEQ ID NO: 5, having undergone a mutation selected from Arg105Gln, and Gln108Leu, or There is provided a multivalent, preferably divalent or trivalent, polypeptide consisting essentially thereof.

In another preferred embodiment, the present invention provides that the amino acid residue is
Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Gly78Leu, Ala83Arg, Asp85Glu, and Arg105Gln, Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Ap
Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54Asp,
Gly54Asp,
Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Ala83Arg,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Asp85Glu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Arg105Gln,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Asp85Glu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Arg105Gln,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Asp85Glu, and Arg105Gln, or Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, Asp85Glu, and Arg105Gln.
A polyvalent comprising or consisting essentially of at least two (preferably identical) amino acid sequences or Nanobodies® comprising or consisting essentially of SEQ ID NO: 5 , Preferably a divalent or trivalent polypeptide.

In another preferred aspect, the invention provides a method comprising:
a) SEQ ID NO: 60 to SEQ ID NO: 76
b) an amino acid sequence having only one amino acid difference from one of SEQ ID NO: 60 to SEQ ID NO: 76, preferably only two, more preferably only one;
i) the amino acid sequence has glutamine (Gln, Q) at position 105 (the position is determined by Kabat numbering);
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) Comprising or consisting essentially of at least three (preferably identical) amino acid sequences or Nanobodies® selected from amino acid sequences (as defined herein). A multivalent, preferably trivalent, polypeptide is provided.

  In another preferred embodiment, the polypeptide of the invention comprises at least three identical amino acid sequences or Nanobodies® selected from the group consisting of one of SEQ ID NO: 60 to SEQ ID NO: 76, Or essentially from this.

In another preferred aspect, the invention provides a method comprising:
a) SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76 b) SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and An amino acid sequence having only one, preferably two, more preferably one amino acid difference from one of SEQ ID NO: 76,
i) the amino acid sequence has glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and / or arginine (Arg, R) at position 83 (the above positions are determined by Kabat numbering; Desired),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) Comprising or consisting essentially of at least three (preferably identical) amino acid sequences or Nanobodies® selected from amino acid sequences (as defined herein). A multivalent, preferably trivalent, polypeptide is provided.

In another preferred aspect, the invention provides a method comprising:
a) SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76
b) only three, preferably only two and more preferably only one amino acid difference from one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75 and SEQ ID NO: 76 An amino acid sequence having
i) the amino acid sequence has glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and arginine (Arg, R) at position 83 (the above positions are determined by Kabat numbering) ),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) Comprising or consisting essentially of at least three (preferably identical) amino acid sequences or Nanobodies® selected from amino acid sequences (as defined herein). A multivalent, preferably trivalent, polypeptide is provided.

  In another preferred aspect, the polypeptide of the present invention comprises at least three of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76. Contain or consist essentially of the same amino acid sequence or Nanobody®.

In another preferred aspect, the invention provides a method comprising:
a) SEQ ID NO: 65 and SEQ ID NO: 76,
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NO: 65 and SEQ ID NO: 76,
i) the amino acid sequence is aspartic acid (Asp, D) at position 54, glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and / or arginine (Arg, R) at position 83; (The position is determined by Kabat numbering),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) Comprising or consisting essentially of at least three (preferably identical) amino acid sequences or Nanobodies® selected from amino acid sequences (as defined herein). A multivalent, preferably trivalent, polypeptide is provided.

In another preferred aspect, the invention provides a method comprising:
a) SEQ ID NO: 65 and SEQ ID NO: 76,
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NO: 65 and SEQ ID NO: 76,
i) the amino acid sequence is aspartic acid (Asp, D) at position 54, glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and / or arginine (Arg, R) at position 83; (The position is determined by Kabat numbering),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) Comprising or consisting essentially of at least three (preferably identical) amino acid sequences or Nanobodies® selected from amino acid sequences (as defined herein). A multivalent, preferably trivalent, polypeptide is provided.

  In another preferred embodiment, the polypeptide of the invention comprises or consists essentially of at least three identical amino acid sequences or Nanobodies® selected from one of SEQ ID NO: 65 and SEQ ID NO: 76. Become

The present invention
a) SEQ ID NO: 146 to SEQ ID NO: 153
b) an amino acid sequence having only one, preferably two, more preferably only one amino acid difference from one of SEQ ID NOs: 146 to 153,
i) the amino acid sequence comprises proline (Pro, P) at position 14, arginine (Arg, R) at position 19, leucine (Leu, L) at position 20 and / or leucine (Leu, L) at position 108 Have (the above positions are determined by Kabat numbering),
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Comprising or consisting essentially of at least three (preferably identical) amino acid sequences or Nanobodies® selected from amino acid sequences (as defined herein); Also provided are multivalent, preferably trivalent, polypeptides.

  In another preferred aspect, the polypeptide of the invention comprises or consists essentially of at least three identical amino acid sequences or Nanobodies® selected from one of SEQ ID NOs: 146-153. Become

In another preferred embodiment, the present invention provides a method comprising: a) SEQ ID NOs: 146-149 and SEQ ID NOs: 151-153;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153,
i) the amino acid sequence comprises proline (Pro, P) at position 14, arginine (Arg, R) at position 19, leucine (Leu, L) at position 20 and / or leucine (Leu, L) at position 108; Arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85, and / or glutamine (Gln, Q) at position 105;
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) Comprising or consisting essentially of at least three (preferably identical) amino acid sequences or Nanobodies® selected from amino acid sequences (as defined herein). A multivalent, preferably trivalent, polypeptide is provided.

In another preferred aspect, the invention provides a method comprising:
a) SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153,
i) the amino acid sequence is
SEQ ID NO: 146 (this amino acid sequence preferably has glutamine (Gln, Q) at position 105);
SEQ ID NO: 147 (this amino acid sequence preferably has arginine (Arg, R) at position 83 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 148 (this amino acid sequence preferably has glutamic acid (Glu, E) at position 85 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 149 (this amino acid sequence preferably has arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 151 (this amino acid sequence preferably has arginine (Arg, R) at position 83);
SEQ ID NO: 152 (this amino acid sequence preferably has glutamic acid (Glu, E) at position 85);
SEQ ID NO: 153 (this amino acid sequence preferably has arginine (Arg, R) at position 83 and glutamic acid (Glu, E) at position 85)
The amino acid sequence has a proline (Pro, P) at position 14 and an arginine (Arg, R) at position 19, as in the case of having only 3, preferably 2, and more preferably only 1 amino acid difference. Leucine (Leu, L) at position 20 and / or leucine (Leu, L) at position 108, arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85, and / or position 105 Has glutamine (Gln, Q) (the position is determined by Kabat numbering),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) Comprising or consisting essentially of at least three (preferably identical) amino acid sequences or Nanobodies® selected from amino acid sequences (as defined herein). A multivalent, preferably trivalent, polypeptide is provided.

  Preferred polypeptides of the invention comprise, or consist essentially of, at least three identical amino acid sequences or Nanobodies® selected from SEQ ID NOs: 146-149 and SEQ ID NOs: 151-153. Become.

  In another preferred aspect, the invention provides one or more (eg, 2, 3, 4, 5, 6, 7, 8, 9, 9, 10, 11, or 12) Has a mutation selected from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and a mutation selected from the sequence consisting of 5 or any of the following: A multivalent, preferably trivalent, polypeptide comprising or consisting essentially of at least three (preferably identical) amino acid sequences or Nanobodies®.

  In another preferred embodiment, the present invention provides that one or more (eg, 2, 3, 4, 5, 6, 7, 8, or 9) amino acid residues are Val5Leu, Ala14Pro, At least three (preferably identical) amino acid sequences comprising or consisting essentially of SEQ ID NO: 5, or having a mutation selected from Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp. There is provided a multivalent, preferably trivalent, polypeptide comprising or consisting essentially of Nanobody®.

  In another preferred embodiment, the invention provides a method wherein the one or more (eg, two, three or four) amino acid residues are mutated wherein the amino acid residues are selected from Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu. Polyvalent, preferably trivalent, polypeptide comprising or consisting essentially of at least three (preferably identical) amino acid sequences or Nanobodies® comprising or consisting essentially of No. 5 I will provide a.

  In another preferred embodiment, the invention provides that one or more (eg, 2, 3, 4, 5, 6, or 7) amino acid residues are Ala14Pro, Ser19Arg, Ile20Leu, Ala83Arg, Asp85Glu, Comprising at least three (preferably identical) amino acid sequences or Nanobodies® comprising or consisting essentially of SEQ ID NO: 5, having undergone a mutation selected from Arg105Gln, and Gln108Leu, or There is provided a multivalent, preferably trivalent, polypeptide consisting essentially of this.

In another preferred embodiment, the present invention provides that the amino acid residue is
Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Gly78Leu, Ala83Arg, Asp85Glu, and Arg105Gln,
Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54Asp,
Gly54Asp,
Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Ala83Arg,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Asp85Glu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Arg105Gln,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Asp85Glu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Arg105Gln,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Asp85Glu, and Arg105Gln, or Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, Asp85Glu, and Arg105Gln.
Multivalent comprising or consisting essentially of at least three (preferably identical) amino acid sequences or Nanobodies® comprising or consisting essentially of SEQ ID NO: 5 , Preferably a trivalent polypeptide.

  The invention also provides a polyvalent, preferably divalent or trivalent, polypeptide as described above, wherein the glutamic acid at position 1 has been changed to aspartic acid.

In this regard, the present invention provides
a) SEQ ID NO: 138 to SEQ ID NO: 141 and SEQ ID NO: 154 to SEQ ID NO: 157;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 138 to 141 and 154 to 157,
i) the amino acid sequence has aspartic acid (Asp, D) at position 1 (the position is determined by Kabat numbering);
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) A polyvalent, preferably bivalent, preferably comprising or consisting essentially of at least one amino acid sequence or Nanobody® selected from amino acid sequences (as defined herein). A multivalent or trivalent polypeptide is provided.

  In another preferred embodiment, the polypeptide of the invention has at least one amino acid sequence or Nanobody® selected from one of SEQ ID NOs: 138-141 and SEQ ID NOs: 154-157. Include or consist essentially of.

  In another preferred embodiment, the present invention relates to a composition comprising at least one amino acid sequence or Nanobody® comprising, or consisting essentially of, SEQ ID NO: 5 wherein glutamic acid at position 1 has been changed to aspartic acid. Or a multivalent, preferably divalent or trivalent, polypeptide consisting essentially of the same.

  In another preferred embodiment, the present invention comprises a sequence comprising at least one amino acid sequence or Nanobody® comprising, or consisting essentially of, SEQ ID NO: 62 wherein glutamic acid at position 1 has been changed to aspartic acid. Or a multivalent, preferably divalent or trivalent, polypeptide consisting essentially of the same.

  In another preferred embodiment, the present invention comprises a sequence comprising at least one amino acid sequence or Nanobody® comprising or consisting essentially of SEQ ID NO: 65 wherein glutamic acid at position 1 has been changed to aspartic acid. Or a multivalent, preferably divalent or trivalent, polypeptide consisting essentially of the same.

  In another preferred embodiment, the present invention relates to a composition comprising at least one amino acid sequence or Nanobody® comprising, or consisting essentially of SEQ ID NO: 76, wherein glutamic acid at position 1 has been changed to aspartic acid. Or a multivalent, preferably divalent or trivalent, polypeptide consisting essentially of the same.

  In another preferred embodiment, the present invention relates to a composition comprising at least one amino acid sequence or Nanobody® comprising, or consisting essentially of SEQ ID NO: 75, wherein glutamic acid at position 1 has been changed to aspartic acid. Or a multivalent, preferably divalent or trivalent, polypeptide consisting essentially of the same.

  In another preferred embodiment, the present invention comprises a SEQ ID NO: 147 wherein glutamic acid at position 1 has been changed to aspartic acid, or comprises at least one amino acid sequence or Nanobody® consisting essentially thereof. Or a multivalent, preferably divalent or trivalent, polypeptide consisting essentially of the same.

  In another preferred embodiment, the present invention comprises or comprises at least one amino acid sequence or Nanobody (R) comprising, or consisting essentially of, SEQ ID NO: 149 wherein glutamic acid at position 1 has been changed to aspartic acid. Or a multivalent, preferably divalent or trivalent, polypeptide consisting essentially of the same.

  In another preferred embodiment, the present invention comprises a SEQ ID NO: 153 wherein glutamic acid at position 1 is changed to aspartic acid, or comprises at least one amino acid sequence or Nanobody® consisting essentially thereof. Or a multivalent, preferably divalent or trivalent, polypeptide consisting essentially of the same.

  In another preferred aspect, the invention provides one or more (eg, 2, 3, 4, 5, 6, 7, 8, 9, 9, 10, 11, or 12) Has a mutation in which the amino acid residue is selected from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp. A polyvalent, preferably divalent or trivalent, comprising or consisting essentially of at least one amino acid sequence or Nanobody® comprising or consisting essentially of SEQ ID NO: 5 A polypeptide is provided.

  In another preferred embodiment, the present invention provides that one or more (eg, 2, 3, 4, 5, 6, 7, 8, or 9) amino acid residues are Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and a mutation selected from Gly54Asp, wherein the glutamic acid at position 1 has been changed to aspartic acid, comprising or essentially consisting of SEQ ID NO: 5. A multivalent, preferably divalent or trivalent, polypeptide comprising or consisting essentially of at least one amino acid sequence or Nanobody®.

  In another preferred aspect, the invention provides that one or more (eg, two, three or four) amino acid residues have a mutation selected from Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu, Wherein the glutamic acid in position 1 has been changed to aspartic acid, comprising or consisting essentially of SEQ ID NO: 5, comprising or consisting essentially of at least one amino acid sequence or Nanobody® , Preferably a divalent or trivalent polypeptide.

  In another preferred embodiment, the invention provides that one or more (e.g., 2, 3, 4, 5, 6, or 7) amino acid residues are Ala14Pro, Ser19Arg, Ile20Leu, Ala83Arg, Asp85Glu, At least one amino acid sequence or Nanobody comprising a mutation consisting of Arg105Gln and Gln108Leu, wherein glutamic acid at position 1 has been changed to aspartic acid, comprising or consisting essentially of SEQ ID NO: 5 ( (Registered trademark) or consists essentially of the same.

In another preferred embodiment, the present invention provides that the amino acid residue is
Glu1Asp,
Glu1Asp, Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Glu1Asp, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Glu1Asp, Gly78Leu, Ala83Arg, Asp85Glu, and Arg105Gln;
Glu1Asp, Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Glu1Asp, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Glu1Asp, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54Asp,
Glu1Asp and Gly54Asp,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Ala83Arg;
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Asp85Glu,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Arg105Gln,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Asp85Glu,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Arg105Gln, Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Asp85Glu, and Arg105Gln, or Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, Asp85Glu, and Arg105Gln
Comprising, or consisting essentially of, SEQ ID NO: 5, comprising or consisting essentially of at least one amino acid sequence or Nanobody®, preferably divalent or divalent. A trivalent polypeptide is provided.

In another preferred aspect, the invention provides a method comprising:
a) SEQ ID NO: 60 to SEQ ID NO: 76
b) an amino acid sequence having only one amino acid difference from one of SEQ ID NO: 60 to SEQ ID NO: 76, preferably only two, more preferably only one;
i) the amino acid sequence has glutamine (Gln, Q) at position 105 (the position is determined by Kabat numbering);
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) A divalent amino acid sequence comprising or consisting essentially of two (preferably identical) amino acid sequences or Nanobodies® selected from amino acid sequences (as defined herein). A polypeptide is provided.

  In another preferred embodiment, the polypeptide of the invention is a bivalent polypeptide, comprising two identical amino acid sequences or Nanobodies® selected from one of SEQ ID NO: 60-SEQ ID NO: 76. Or consisting essentially of:

In another preferred aspect, the invention provides a method comprising:
a) SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76
b) only three, preferably only two and more preferably only one amino acid difference from one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75 and SEQ ID NO: 76 An amino acid sequence having
i) the amino acid sequence has glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and / or arginine (Arg, R) at position 83 (the above positions are determined by Kabat numbering; Desired),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) A divalent amino acid sequence comprising or consisting essentially of two (preferably identical) amino acid sequences or Nanobodies® selected from amino acid sequences (as defined herein). A polypeptide is provided.

In another preferred aspect, the invention provides a method comprising:
a) SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76
b) only three, preferably only two and more preferably only one amino acid difference from one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75 and SEQ ID NO: 76 An amino acid sequence having
i) the amino acid sequence has glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and arginine (Arg, R) at position 83 (the above positions are determined by Kabat numbering) ),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) A divalent amino acid sequence comprising or consisting essentially of two (preferably identical) amino acid sequences or Nanobodies® selected from amino acid sequences (as defined herein). A polypeptide is provided.

  In another preferred aspect, the polypeptide of the invention is a bivalent polypeptide, selected from one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76. , Or consists essentially of two identical amino acid sequences or Nanobodies®.

In another preferred aspect, the invention provides a method comprising:
a) SEQ ID NO: 65 and SEQ ID NO: 76,
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NO: 65 and SEQ ID NO: 76,
i) the amino acid sequence is aspartic acid (Asp, D) at position 54, glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and / or arginine (Arg, R) at position 83; (The position is determined by Kabat numbering),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) A divalent amino acid sequence comprising or consisting essentially of two (preferably identical) amino acid sequences or Nanobodies® selected from amino acid sequences (as defined herein). A polypeptide is provided.

In another preferred aspect, the invention provides a method comprising:
a) SEQ ID NO: 65 and SEQ ID NO: 76,
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NO: 65 and SEQ ID NO: 76,
i) the amino acid sequence has aspartic acid (Asp, D) at position 54, glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and arginine (Arg, R) at position 83; (The above position is obtained by Kabat numbering)
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) A divalent amino acid sequence comprising or consisting essentially of two (preferably identical) amino acid sequences or Nanobodies® selected from amino acid sequences (as defined herein). A polypeptide is provided.

  In another preferred embodiment, the polypeptide of the invention is a bivalent polypeptide, comprising or consisting essentially of two identical amino acid sequences or Nanobodies® selected from SEQ ID NO: 65 and SEQ ID NO: 76. Become

The present invention
a) SEQ ID NO: 146 to SEQ ID NO: 153
b) an amino acid sequence having only one, preferably two, more preferably only one amino acid difference from one of SEQ ID NOs: 146 to 153,
i) the amino acid sequence comprises proline (Pro, P) at position 14, arginine (Arg, R) at position 19, leucine (Leu, L) at position 20 and / or leucine (Leu, L) at position 108 Have (the above positions are determined by Kabat numbering),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) A divalent amino acid sequence comprising or consisting essentially of two (preferably identical) amino acid sequences or Nanobodies® selected from amino acid sequences (as defined herein). Also provided are polypeptides.

  In another preferred embodiment, the polypeptide of the invention is a bivalent polypeptide and comprises two identical amino acid sequences or Nanobodies® selected from one of SEQ ID NO: 146-153. Or consisting essentially of:

In another preferred aspect, the invention provides a method comprising:
a) SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153,
i) the amino acid sequence comprises proline (Pro, P) at position 14, arginine (Arg, R) at position 19, leucine (Leu, L) at position 20, and / or leucine (Leu, L) at position 108 Having arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85, and / or glutamine (Gln, Q) at position 105;
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) A divalent amino acid sequence comprising or consisting essentially of two (preferably identical) amino acid sequences or Nanobodies® selected from amino acid sequences (as defined herein). A polypeptide is provided.

In another preferred aspect, the invention provides a method comprising:
a) SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153,
i) the amino acid sequence is
SEQ ID NO: 146 (this amino acid sequence preferably has glutamine (Gln, Q) at position 105); SEQ ID NO: 147 (This amino acid sequence preferably has arginine (Arg, R) at position 83 and glutamine (Gln, Q)),
SEQ ID NO: 148 (this amino acid sequence preferably has glutamic acid (Glu, E) at position 85 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 149 (this amino acid sequence preferably has arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 151 (this amino acid sequence preferably has arginine (Arg, R) at position 83);
SEQ ID NO: 152 (this amino acid sequence preferably has glutamic acid (Glu, E) at position 85);
SEQ ID NO: 153 (this amino acid sequence preferably has arginine (Arg, R) at position 83 and glutamic acid (Glu, E) at position 85)
The amino acid sequence has a proline (Pro, P) at position 14 and an arginine (Arg, R) at position 19, as in the case of having only 3, preferably 2, and more preferably only 1 amino acid difference. Leucine (Leu, L) at position 20 and / or leucine (Leu, L) at position 108, arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85, and / or position 105 Has glutamine (Gln, Q) (the position is determined by Kabat numbering),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) A divalent amino acid sequence comprising or consisting essentially of two (preferably identical) amino acid sequences or Nanobodies® selected from amino acid sequences (as defined herein). A polypeptide is provided.

  Preferred polypeptides of the invention comprise or consist essentially of two amino acid sequences or Nanobodies® selected from SEQ ID NOs: 146-149 and 151-153.

  In another preferred aspect, the invention provides one or more (eg, 2, 3, 4, 5, 6, 7, 8, 9, 9, 10, 11, or 12) Has a mutation selected from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and a mutation selected from the sequence consisting of 5 or any of the following: A bivalent polypeptide comprising or consisting essentially of two (preferably identical) amino acid sequences or Nanobodies®.

  In another preferred embodiment, the present invention provides that one or more (eg, 2, 3, 4, 5, 6, 7, 8, or 9) amino acid residues are Val5Leu, Ala14Pro, Two (preferably identical) amino acid sequences or Nanobodies comprising or consisting essentially of SEQ ID NO: 5, mutated selected from Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp. (Registered trademark) or a bivalent polypeptide consisting essentially of the same.

  In another preferred embodiment, the invention provides a method wherein the one or more (eg, two, three or four) amino acid residues are mutated wherein the amino acid residues are selected from Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu. There is provided a bivalent polypeptide comprising or consisting essentially of two (preferably identical) amino acid sequences or Nanobodies® comprising or consisting essentially of No. 5.

  In another preferred embodiment, the invention provides that one or more (e.g., 2, 3, 4, 5, 6, or 7) amino acid residues are Ala14Pro, Ser19Arg, Ile20Leu, Ala83Arg, Asp85Glu, Contains or consists of two (preferably identical) amino acid sequences or Nanobodies® comprising or consisting essentially of SEQ ID NO: 5, having undergone a mutation selected from Arg105Gln, and Gln108Leu. An essentially bivalent polypeptide is provided.

In another preferred embodiment, the present invention provides that the amino acid residue is
Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Gly78Leu, Ala83Arg, Asp85Glu, and Arg105Gln,
Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54Asp,
Gly54Asp,
Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Ala83Arg,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Asp85Glu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Arg105Gln,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Asp85Glu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Arg105Gln,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Asp85Glu, and Arg105Gln, or Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, Asp85Glu, and Arg105Gln.
A divalent polymorph comprising, or consisting essentially of, two (preferably identical) amino acid sequences or Nanobodies® comprising or consisting essentially of SEQ ID NO: 5 Provide a peptide.

  The present invention also provides a divalent polypeptide as described above, wherein the glutamic acid at position 1 has been changed to aspartic acid.

In this regard, the present invention provides
a) SEQ ID NO: 138 to SEQ ID NO: 141 and SEQ ID NO: 154 to SEQ ID NO: 157, b) One of SEQ ID NO: 138 to SEQ ID NO: 141 and SEQ ID NO: 154 to SEQ ID NO: 157 and only three, preferably only two, More preferably an amino acid sequence having only one amino acid difference,
i) the amino acid sequence has aspartic acid (Asp, D) at position 1 (the position is determined by Kabat numbering);
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) Also provided is a bivalent polypeptide comprising or consisting essentially of at least one amino acid sequence or Nanobody® selected from an amino acid sequence (as defined herein). .

  In another preferred embodiment, the polypeptide of the invention has at least one amino acid sequence or Nanobody® selected from one of SEQ ID NOs: 138-141 and SEQ ID NOs: 154-157. Include or consist essentially of.

  In another preferred embodiment, the present invention relates to a composition comprising at least one amino acid sequence or Nanobody® comprising, or consisting essentially of, SEQ ID NO: 5 wherein glutamic acid at position 1 has been changed to aspartic acid. Or a bivalent polypeptide consisting essentially of the same.

  In another preferred embodiment, the present invention comprises a sequence comprising at least one amino acid sequence or Nanobody® comprising, or consisting essentially of, SEQ ID NO: 62 wherein glutamic acid at position 1 has been changed to aspartic acid. Or a bivalent polypeptide consisting essentially of the same.

  In another preferred embodiment, the present invention comprises a sequence comprising at least one amino acid sequence or Nanobody® comprising or consisting essentially of SEQ ID NO: 65 wherein glutamic acid at position 1 has been changed to aspartic acid. Or a bivalent polypeptide consisting essentially of the same.

  In another preferred embodiment, the present invention relates to a composition comprising at least one amino acid sequence or Nanobody® comprising, or consisting essentially of SEQ ID NO: 76, wherein glutamic acid at position 1 has been changed to aspartic acid. Or a bivalent polypeptide consisting essentially of the same.

  In another preferred embodiment, the present invention relates to a composition comprising at least one amino acid sequence or Nanobody® comprising, or consisting essentially of SEQ ID NO: 75, wherein glutamic acid at position 1 has been changed to aspartic acid. Or a bivalent polypeptide consisting essentially of the same.

  In another preferred embodiment, the present invention comprises a SEQ ID NO: 147 wherein glutamic acid at position 1 has been changed to aspartic acid, or comprises at least one amino acid sequence or Nanobody® consisting essentially thereof. Or a bivalent polypeptide consisting essentially of the same.

  In another preferred embodiment, the present invention comprises or comprises at least one amino acid sequence or Nanobody (R) comprising, or consisting essentially of, SEQ ID NO: 149 wherein glutamic acid at position 1 has been changed to aspartic acid. Or a bivalent polypeptide consisting essentially of the same.

  In another preferred embodiment, the present invention comprises a SEQ ID NO: 153 wherein glutamic acid at position 1 is changed to aspartic acid, or comprises at least one amino acid sequence or Nanobody® consisting essentially thereof. Or a bivalent polypeptide consisting essentially of the same.

  In another preferred aspect, the invention provides one or more (eg, 2, 3, 4, 5, 6, 7, 8, 9, 9, 10, 11, or 12) Has a mutation in which the amino acid residue is selected from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp. Or a divalent polypeptide comprising or consisting essentially of at least one amino acid sequence or Nanobody®, comprising or consisting essentially of SEQ ID NO: 5.

  In another preferred embodiment, the present invention provides that one or more (eg, 2, 3, 4, 5, 6, 7, 8, or 9) amino acid residues are Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and a mutation selected from Gly54Asp, wherein the glutamic acid at position 1 has been changed to aspartic acid, comprising or essentially consisting of SEQ ID NO: 5. A bivalent polypeptide comprising or consisting essentially of at least one amino acid sequence or Nanobody®.

  In another preferred aspect, the invention provides that one or more (eg, two, three or four) amino acid residues have a mutation selected from Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu, Wherein the glutamic acid in position 1 has been changed to aspartic acid, comprising or consisting essentially of SEQ ID NO: 5, comprising or consisting essentially of at least one amino acid sequence or Nanobody® A polypeptide is provided.

  In another preferred embodiment, the invention provides that one or more (eg, 2, 3, 4, 5, 6, or 7) amino acid residues are Ala14Pro, Ser19Arg, Ile20Leu, Ala83Arg, Asp85Glu, At least one amino acid sequence or Nanobody comprising a mutation consisting of Arg105Gln and Gln108Leu, wherein glutamic acid at position 1 has been changed to aspartic acid, comprising or consisting essentially of SEQ ID NO: 5 ( (Registered Trademark) is provided.

In another preferred embodiment, the present invention provides that the amino acid residue is
Glu1Asp,
Glu1Asp, Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Glu1Asp, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Glu1Asp, Gly78Leu, Ala83Arg, Asp85Glu, and Arg105Gln;
Glu1Asp, Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Glu1Asp, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Glu1Asp, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54Asp,
Glu1Asp and Gly54Asp,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Ala83Arg;
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Asp85Glu,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Arg105Gln,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Asp85Glu,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Arg105Gln,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Asp85Glu, and Arg105Gln, or Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Ag, ArA85Ag, ArA85Ag, ArA85Ag
A bivalent polypeptide comprising or consisting essentially of at least one amino acid sequence or Nanobody®, comprising or consisting essentially of SEQ ID NO: 5.

In another preferred aspect, the invention provides a method comprising:
a) SEQ ID NO: 60 to SEQ ID NO: 76
b) an amino acid sequence having only one amino acid difference from one of SEQ ID NO: 60 to SEQ ID NO: 76, preferably only two, more preferably only one;
i) the amino acid sequence has glutamine (Gln, Q) at position 105 (the position is determined by Kabat numbering);
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) A trivalent sequence comprising or consisting essentially of three (preferably identical) amino acid sequences or Nanobodies® selected from amino acid sequences (as defined herein). A polypeptide is provided.

  In another preferred embodiment, the polypeptide of the invention is a trivalent polypeptide and comprises three identical amino acid sequences or Nanobodies® selected from one of SEQ ID NO: 60-SEQ ID NO: 76. Or consisting essentially of:

In another preferred aspect, the invention provides a method comprising:
a) SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76
b) only three, preferably only two and more preferably only one amino acid difference from one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75 and SEQ ID NO: 76 An amino acid sequence having
i) the amino acid sequence has glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and / or arginine (Arg, R) at position 83 (the above positions are determined by Kabat numbering; Desired),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) A trivalent sequence comprising or consisting essentially of three (preferably identical) amino acid sequences or Nanobodies® selected from amino acid sequences (as defined herein). A polypeptide is provided.

In another preferred aspect, the invention provides a method comprising:
a) SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76
b) only three, preferably only two and more preferably only one amino acid difference from one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75 and SEQ ID NO: 76 An amino acid sequence having
i) the amino acid sequence has glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and arginine (Arg, R) at position 83 (the above positions are determined by Kabat numbering) ),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) A trivalent sequence comprising or consisting essentially of three (preferably identical) amino acid sequences or Nanobodies® selected from amino acid sequences (as defined herein). A polypeptide is provided.

  In another preferred aspect, the polypeptide of the invention is a trivalent polypeptide, selected from one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76. Comprise or consist essentially of three identical amino acid sequences or Nanobodies®

In another preferred aspect, the invention provides a method comprising:
a) SEQ ID NO: 65 and SEQ ID NO: 76,
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NO: 65 and SEQ ID NO: 76,
i) the amino acid sequence is aspartic acid (Asp, D) at position 54, glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and / or arginine (Arg, R) at position 83; (The position is determined by Kabat numbering),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) A trivalent sequence comprising or consisting essentially of three (preferably identical) amino acid sequences or Nanobodies® selected from amino acid sequences (as defined herein). A polypeptide is provided.

In another preferred aspect, the invention provides a method comprising:
a) SEQ ID NO: 65 and SEQ ID NO: 76,
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NO: 65 and SEQ ID NO: 76,
i) the amino acid sequence has aspartic acid (Asp, D) at position 54, glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and arginine (Arg, R) at position 83; (The above position is obtained by Kabat numbering)
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) A trivalent sequence comprising or consisting essentially of three (preferably identical) amino acid sequences or Nanobodies® selected from amino acid sequences (as defined herein). A polypeptide is provided.

  In another preferred embodiment, the polypeptide of the invention is a trivalent polypeptide, comprising three identical amino acid sequences or Nanobodies® selected from one of SEQ ID NO: 65 and SEQ ID NO: 76. Or consisting essentially of:

The present invention
a) SEQ ID NO: 146 to SEQ ID NO: 153
b) an amino acid sequence having only one, preferably two, more preferably only one amino acid difference from one of SEQ ID NOs: 146 to 153,
i) the amino acid sequence comprises proline (Pro, P) at position 14, arginine (Arg, R) at position 19, leucine (Leu, L) at position 20 and / or leucine (Leu, L) at position 108 Have (the above positions are determined by Kabat numbering),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) A trivalent sequence comprising or consisting essentially of three (preferably identical) amino acid sequences or Nanobodies® selected from amino acid sequences (as defined herein). Also provided are polypeptides.

  In another preferred embodiment, the polypeptide of the present invention is a trivalent polypeptide, comprising three identical amino acid sequences or Nanobodies® selected from one of SEQ ID NO: 146 to SEQ ID NO: 153. Or consisting essentially of:

In another preferred aspect, the invention provides a method comprising:
a) SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153,
i) the amino acid sequence comprises proline (Pro, P) at position 14, arginine (Arg, R) at position 19, leucine (Leu, L) at position 20, and / or leucine (Leu, L) at position 108 Having arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85, and / or glutamine (Gln, Q) at position 105;
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) A trivalent sequence comprising or consisting essentially of three (preferably identical) amino acid sequences or Nanobodies® selected from amino acid sequences (as defined herein). A polypeptide is provided.

In another preferred aspect, the invention provides a method comprising:
a) SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153,
i) the amino acid sequence is
SEQ ID NO: 146 (this amino acid sequence preferably has glutamine (Gln, Q) at position 105);
SEQ ID NO: 147 (this amino acid sequence preferably has arginine (Arg, R) at position 83 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 148 (this amino acid sequence preferably has glutamic acid (Glu, E) at position 85 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 149 (this amino acid sequence preferably has arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 151 (this amino acid sequence preferably has arginine (Arg, R) at position 83);
SEQ ID NO: 152 (this amino acid sequence preferably has glutamic acid (Glu, E) at position 85);
SEQ ID NO: 153 (this amino acid sequence preferably has arginine (Arg, R) at position 83 and glutamic acid (Glu, E) at position 85)
The amino acid sequence has a proline (Pro, P) at position 14 and an arginine (Arg, R) at position 19, as in the case of having only 3, preferably 2, and more preferably only 1 amino acid difference. Leucine (Leu, L) at position 20 and / or leucine (Leu, L) at position 108, arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85, and / or position 105 Has glutamine (Gln, Q) (the position is determined by Kabat numbering),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) A trivalent sequence comprising or consisting essentially of three (preferably identical) amino acid sequences or Nanobodies® selected from amino acid sequences (as defined herein). A polypeptide is provided.

  Preferred polypeptides of the invention comprise or consist essentially of three amino acid sequences or Nanobodies® selected from SEQ ID NOs: 146-149 and SEQ ID NOs: 151-153.

  In another preferred aspect, the invention provides one or more (eg, 2, 3, 4, 5, 6, 7, 8, 9, 9, 10, 11, or 12) Has a mutation selected from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and a mutation selected from the sequence consisting of 5 or any of the following: A trivalent polypeptide comprising or consisting essentially of three (preferably identical) amino acid sequences or Nanobodies®.

  In another preferred embodiment, the present invention provides that one or more (eg, 2, 3, 4, 5, 6, 7, 8, or 9) amino acid residues are Val5Leu, Ala14Pro, Three (preferably identical) amino acid sequences or Nanobodies comprising or consisting essentially of SEQ ID NO: 5, mutated from Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp. (Registered Trademark) is provided.

  In another preferred embodiment, the invention provides a method wherein the one or more (eg, two, three or four) amino acid residues are mutated wherein the amino acid residues are selected from Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu. There is provided a trivalent polypeptide comprising or consisting essentially of three (preferably identical) amino acid sequences or Nanobodies® comprising or consisting essentially of No. 5.

  In another preferred embodiment, the invention provides that one or more (e.g., 2, 3, 4, 5, 6, or 7) amino acid residues are Ala14Pro, Ser19Arg, Ile20Leu, Ala83Arg, Asp85Glu, Contains or consists of three (preferably identical) amino acid sequences or Nanobodies® comprising or consisting essentially of SEQ ID NO: 5, having undergone a mutation selected from Arg105Gln, and Gln108Leu. An essentially trivalent polypeptide is provided.

In another preferred embodiment, the present invention provides that the amino acid residue is
Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Gly78Leu, Ala83Arg, Asp85Glu, and Arg105Gln,
Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54Asp,
Gly54Asp,
Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Ala83Arg,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Asp85Glu, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Arg105Gln,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Asp85Glu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Arg105Gln,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Asp85Glu, and Arg105Gln, or Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, Asp85Glu, and Arg105Gln.
A trivalent polymorph comprising or consisting essentially of three (preferably identical) amino acid sequences or Nanobodies® comprising or consisting essentially of SEQ ID NO: 5 Provide a peptide.

  Preferred multivalent polypeptides of the invention comprise or consist essentially of the three amino acid sequences having the SEQ ID NO: 62 or Nanobodies®. Another preferred multivalent polypeptide of the invention comprises or consists essentially of the three amino acid sequence having SEQ ID NO: 65 or Nanobody®. Another preferred multivalent polypeptide of the present invention comprises or consists essentially of the three amino acid sequence having SEQ ID NO: 76 or Nanobody®. Another preferred multivalent polypeptide of the invention comprises or consists essentially of the three amino acid sequence or Nanobody® having SEQ ID NO: 75. Another preferred multivalent polypeptide of the invention comprises or consists essentially of the three amino acid sequence or Nanobody® having SEQ ID NO: 147. Another preferred multivalent polypeptide of the present invention comprises or consists essentially of the three amino acid sequence or Nanobody® having SEQ ID NO: 149. Another preferred multivalent polypeptide of the invention comprises or consists essentially of the three amino acid sequences having SEQ ID NO: 153 or Nanobodies®.

  The present invention also provides a trivalent polypeptide as described above, wherein the glutamic acid at position 1 has been changed to aspartic acid.

In this regard, the present invention provides
a) SEQ ID NO: 138 to SEQ ID NO: 141 and SEQ ID NO: 154 to SEQ ID NO: 157;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 138 to 141 and 154 to 157,
i) the amino acid sequence has aspartic acid (Asp, D) at position 1 (the position is determined by Kabat numbering);
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) Also provided is a trivalent polypeptide comprising or consisting essentially of at least one amino acid sequence or Nanobody® selected from the amino acid sequences (as defined herein). .

  In another preferred embodiment, the polypeptide of the invention has at least one amino acid sequence or Nanobody® selected from one of SEQ ID NOs: 138-141 and SEQ ID NOs: 154-157. Include or consist essentially of.

  In another preferred embodiment, the present invention relates to a composition comprising at least one amino acid sequence or Nanobody® comprising, or consisting essentially of, SEQ ID NO: 5 wherein glutamic acid at position 1 has been changed to aspartic acid. Or a trivalent polypeptide consisting essentially of the same.

  In another preferred embodiment, the present invention comprises a sequence comprising at least one amino acid sequence or Nanobody® comprising, or consisting essentially of, SEQ ID NO: 62 wherein glutamic acid at position 1 has been changed to aspartic acid. Or a trivalent polypeptide consisting essentially of the same.

  In another preferred embodiment, the present invention comprises a sequence comprising at least one amino acid sequence or Nanobody® comprising or consisting essentially of SEQ ID NO: 65 wherein glutamic acid at position 1 has been changed to aspartic acid. Or a trivalent polypeptide consisting essentially of the same.

  In another preferred embodiment, the present invention relates to a composition comprising at least one amino acid sequence or Nanobody® comprising, or consisting essentially of SEQ ID NO: 76, wherein glutamic acid at position 1 has been changed to aspartic acid. Or a trivalent polypeptide consisting essentially of the same.

  In another preferred embodiment, the present invention relates to a composition comprising at least one amino acid sequence or Nanobody® comprising, or consisting essentially of SEQ ID NO: 75, wherein glutamic acid at position 1 has been changed to aspartic acid. Or a trivalent polypeptide consisting essentially of the same.

  In another preferred embodiment, the present invention comprises a SEQ ID NO: 147 wherein glutamic acid at position 1 has been changed to aspartic acid, or comprises at least one amino acid sequence or Nanobody® consisting essentially thereof. Or a trivalent polypeptide consisting essentially of the same.

  In another preferred embodiment, the present invention comprises or comprises at least one amino acid sequence or Nanobody (R) comprising, or consisting essentially of, SEQ ID NO: 149 wherein glutamic acid at position 1 has been changed to aspartic acid. Or a trivalent polypeptide consisting essentially of the same.

  In another preferred embodiment, the present invention comprises a SEQ ID NO: 153 wherein glutamic acid at position 1 is changed to aspartic acid, or comprises at least one amino acid sequence or Nanobody® consisting essentially thereof. Or a trivalent polypeptide consisting essentially of the same.

  In another preferred aspect, the invention provides one or more (eg, 2, 3, 4, 5, 6, 7, 8, 9, 9, 10, 11, or 12) Has a mutation in which the amino acid residue is selected from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp. Or a trivalent polypeptide comprising or consisting essentially of at least one amino acid sequence or Nanobody® comprising or consisting essentially of SEQ ID NO: 5.

  In another preferred embodiment, the present invention provides that one or more (eg, 2, 3, 4, 5, 6, 7, 8, or 9) amino acid residues are Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and a mutation selected from Gly54Asp, wherein the glutamic acid at position 1 has been changed to aspartic acid, comprising or essentially consisting of SEQ ID NO: 5. Or a trivalent polypeptide comprising or consisting essentially of at least one amino acid sequence or Nanobody®.

  In another preferred aspect, the invention provides that one or more (eg, two, three or four) amino acid residues have a mutation selected from Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu, Wherein the glutamic acid at position 1 is changed to aspartic acid, comprising or consisting essentially of SEQ ID NO: 5, comprising or consisting essentially of at least one amino acid sequence or Nanobody® A polypeptide is provided.

  In another preferred embodiment, the invention provides that one or more (eg, 2, 3, 4, 5, 6, or 7) amino acid residues are Ala14Pro, Ser19Arg, Ile20Leu, Ala83Arg, Asp85Glu, At least one amino acid sequence or Nanobody comprising or consisting essentially of SEQ ID NO: 5, which has a mutation selected from Arg105Gln and Gln108Leu, wherein glutamic acid at position 1 has been changed to aspartic acid ( (Registered Trademark) is provided.

In another preferred embodiment, the present invention provides that the amino acid residue is
Glu1Asp,
Glu1Asp, Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Glu1Asp, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Glu1Asp, Gly78Leu, Ala83Arg, Asp85Glu, and Arg105Gln;
Glu1Asp, Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Glu1Asp, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Glu1Asp, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54Asp,
Glu1Asp and Gly54Asp,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Ala83Arg;
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Asp85Glu,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Arg105Gln,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Asp85Glu, Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Ag, and Arg105Glu
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Asp85Glu, and Arg105Gln, or Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Ag, ArA85Ag, ArA85Ag, ArA85Ag
A trivalent polypeptide comprising or consisting essentially of at least one amino acid sequence or Nanobody®, comprising or consisting essentially of SEQ ID NO: 5 mutated.

In another preferred aspect, the invention provides the following polypeptide:
a) SEQ ID NO: 77 to SEQ ID NO: 79 and SEQ ID NO: 158;
b) a polypeptide which has no more than 3, preferably no more than 2, and more preferably no more than one amino acid difference from one of SEQ ID NOs: 77-79 and 158,
i) the amino acid sequence or Nanobody (registered trademark) contained in the polypeptide is glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, arginine (Arg, R) at position 83; And / or has glutamic acid (Glu, E) at position 85 (the position is determined by Kabat numbering),
ii) the polypeptide has the same, about the same, or a higher affinity as compared to a polypeptide that does not have three, two or one amino acid difference (as the affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or wherein said polypeptide has the same, about the same, or higher potency (as compared to a polypeptide without three, two or one amino acid difference) (As defined herein) is provided.

In a preferred embodiment, the invention provides the following polypeptide:
a) SEQ ID NO: 77 to SEQ ID NO: 79 and SEQ ID NO: 158;
b) a polypeptide which has no more than 3, preferably no more than 2, and more preferably no more than one amino acid difference from one of SEQ ID NOs: 77-79 and 158,
i) the amino acid sequence or Nanobody (registered trademark) contained in the polypeptide is glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, arginine (Arg, R) at position 83; And has glutamic acid (Glu, E) at position 85 (the position is determined by Kabat numbering),
ii) the polypeptide has the same, about the same, or a higher affinity as compared to a polypeptide that does not have three, two or one amino acid difference (as the affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or wherein said polypeptide has the same, about the same, or higher potency (as compared to a polypeptide without three, two or one amino acid difference) (As defined herein) is provided.

  Preferred trivalent polypeptides of the invention comprise or consist essentially of one of SEQ ID NOs: 77-79 and SEQ ID NO: 158.

In another preferred aspect, the invention provides the following polypeptide:
a) SEQ ID NO: 78 and SEQ ID NO: 79,
b) a polypeptide having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NO: 78 and SEQ ID NO: 79,
i) The amino acid sequence or Nanobody (registered trademark) contained in the polypeptide is aspartic acid (Asp, D) at position 54, glutamine (Gln, Q) at position 105, and leucine (Leu, L) at position 78 Having arginine (Arg, R) at position 83 and / or glutamic acid (Glu, E) at position 85 (the position is determined by Kabat numbering);
ii) the polypeptide has the same, about the same, or a higher affinity as compared to a polypeptide that does not have three, two or one amino acid difference (as the affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or wherein said polypeptide has the same, about the same, or higher potency (as compared to a polypeptide without three, two or one amino acid difference) (As defined herein) is provided.

In a preferred embodiment, the invention provides the following polypeptide:
a) a polypeptide having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NO: 78 and SEQ ID NO: 79, or b) one of SEQ ID NO: 78 and SEQ ID NO: 79. ,
i) The amino acid sequence or Nanobody (registered trademark) contained in the polypeptide is aspartic acid (Asp, D) at position 54, glutamine (Gln, Q) at position 105, and leucine (Leu, L) at position 78 Having arginine (Arg, R) at position 83 and glutamic acid (Glu, E) at position 85 (the above positions are determined by Kabat numbering);
ii) the polypeptide has the same, about the same, or a higher affinity as compared to a polypeptide that does not have three, two or one amino acid difference (as the affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or wherein said polypeptide has the same, about the same, or higher potency (as compared to a polypeptide without three, two or one amino acid difference) (As defined herein) is provided.

  Preferred trivalent polypeptides of the invention comprise or consist essentially of SEQ ID NO: 78. Another preferred trivalent polypeptide of the invention comprises or consists essentially of SEQ ID NO: 79.

In another preferred aspect, the invention provides the following polypeptide:
a) SEQ ID NO: 159 to SEQ ID NO: 161,
b) a polypeptide having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 159 to 161;
i) the amino acid sequence or Nanobody (registered trademark) contained in the polypeptide is proline (Pro, P) at position 14, arginine (Arg, R) at position 19, leucine (Leu, L) at position 20, And / or has leucine (Leu, L) at position 108 (the position is determined by Kabat numbering);
ii) the polypeptide has the same, about the same, or a higher affinity as compared to a polypeptide that does not have three, two or one amino acid difference (as the affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or wherein said polypeptide has the same, about the same, or higher potency (as compared to a polypeptide without three, two or one amino acid difference) (As defined herein) is provided.

  Preferred trivalent polypeptides of the invention comprise or consist essentially of one of SEQ ID NOs: 159-161.

In another preferred aspect, the invention provides the following polypeptide:
a) SEQ ID NO: 159 to SEQ ID NO: 161,
b) a polypeptide having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 159 to 161;
i) the amino acid sequence or Nanobody (registered trademark) contained in the polypeptide is proline (Pro, P) at position 14, arginine (Arg, R) at position 19, leucine (Leu, L) at position 20, And / or leucine (Leu, L) at position 108, arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85, and / or glutamine (Gln, Q) at position 105. (The above position is obtained by Kabat numbering)
ii) the polypeptide has the same, about the same, or a higher affinity as compared to a polypeptide that does not have three, two or one amino acid difference (as the affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or wherein said polypeptide has the same, about the same, or higher potency (as compared to a polypeptide without three, two or one amino acid difference) (As defined herein) is provided.

In another preferred aspect, the invention provides the following polypeptide:
a) SEQ ID NO: 159 to SEQ ID NO: 161,
b) a polypeptide having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 159 to 161;
i) the polypeptide is
SEQ ID NO: 159 (amino acid sequence or Nanobody® included in the polypeptide preferably has arginine (Arg, R) at position 83 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 160 (the amino acid sequence or Nanobody® included in the above polypeptide is preferably arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85 and glutamine (Gln, Q)),
SEQ ID NO: 161 (The amino acid sequence or Nanobody® included in the polypeptide preferably has arginine (Arg, R) at position 83 and glutamic acid (Glu, E) at position 85)
And the amino acid sequence or Nanobody (R) encompassed within the polypeptide, as in the case of having only 3, preferably 2, and more preferably only 1 amino acid difference. , P), arginine (Arg, R) at position 19, leucine (Leu, L) at position 20 and / or leucine (Leu, L) at position 108, and arginine (Arg, R), 85 at position 83 Glutamic acid (Glu, E) at position and / or glutamine (Gln, Q) at position 105 (the position is determined by Kabat numbering);
ii) the polypeptide has the same, about the same, or a higher affinity as compared to a polypeptide that does not have three, two or one amino acid difference (as the affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or wherein said polypeptide has the same, about the same, or higher potency (as compared to a polypeptide without three, two or one amino acid difference) (As defined herein) is provided.

  Preferred trivalent polypeptides of the invention comprise or consist essentially of one of SEQ ID NOs: 159-161.

  In another preferred embodiment, the invention relates to at least one (preferably two, more preferably all three) Nanobodies® forming part of SEQ ID NO: 53, wherein one or more (eg 2 , 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) amino acid residues are Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser A trivalent polypeptide comprising or consisting essentially of SEQ ID NO: 53, mutated selected from Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp.

  In another preferred embodiment, the invention relates to at least one (preferably two, more preferably all three) Nanobodies® forming part of SEQ ID NO: 53, wherein one or more (eg 2 , 3, 4, 5, 6, 7, 8, or 9) amino acid residues are selected from Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp. A trivalent polypeptide comprising or consisting essentially of SEQ ID NO: 53, which has been mutated.

  In another preferred embodiment, the invention relates to at least one (preferably two, more preferably all three) Nanobodies® forming part of SEQ ID NO: 53, wherein one or more (eg 2 (3, 4 or 4) amino acid residues comprising a trivalent polypeptide comprising or consisting essentially of SEQ ID NO: 53, mutated selected from Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu. provide.

  In another preferred embodiment, the invention relates to at least one (preferably two, more preferably all three) Nanobodies® forming part of SEQ ID NO: 53, wherein one or more (eg 2 , 3, 4, 5, 6, or 7) amino acid residues have been mutated selected from Ala14Pro, Ser19Arg, Ile20Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu, SEQ ID NO: 53. Or a trivalent polypeptide comprising or consisting essentially of:

In another preferred embodiment, the invention relates to at least one (preferably two, more preferably all three) Nanobodies® forming part of SEQ ID NO: 53, wherein the amino acid residue is:
Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Gly78Leu, Ala83Arg, Asp85Glu, and Arg105Gln,
Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54Asp,
Gly54Asp,
Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Ala83Arg, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Asp85Glu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Arg105Gln,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Asp85Glu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Arg105Gln,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Asp85Glu, and Arg105Gln, or Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, Asp85Glu, and Arg105Gln.
A trivalent polypeptide comprising or consisting essentially of SEQ ID NO: 53, mutated to

  In another preferred embodiment, the polypeptide of the invention consists essentially of the amino acid sequence of SEQ ID NO: 77. In another preferred embodiment, the polypeptide of the invention consists essentially of the amino acid sequence of SEQ ID NO: 78. In another preferred embodiment, the polypeptide of the invention consists essentially of the amino acid sequence of SEQ ID NO: 79. In another preferred aspect, the polypeptide of the invention consists essentially of one of SEQ ID NOs: 158-161.

  In making the polypeptides of the invention, high levels of pyroglutamic acid (pGlu) were detected on the amino terminus by RP-HPLC. After the fermentation, more than 15% of the pGlu level was detected, and during the stability test the pGlu level gradually increased during storage. Such changes cause inhomogeneities in the final product that need to be avoided. Thus, controlling / blocking pGlu formation is important in maintaining therapeutic proteins within their specifications. Certain liquid formulations and / or storage conditions that minimize the formation of pyroglutamic acid are required for proteins with N-terminal glutamate.

  In the present invention, the possibility of N-terminal pGlu post-translational modification by changing N-terminal glutamic acid (E) [HOOC- (CH2) 2-protein] to aspartic acid (D) [HOOC-CH2-protein] Was eliminated and the stability of the product increased. Accordingly, the present invention also relates to a polypeptide as described above, wherein the glutamic acid at position 1 has been changed to aspartic acid.

  The present invention provides a number of sequence optimized polypeptides that show increased stability upon storage during stability testing. Accordingly, the present invention provides the above trivalent polypeptide in which the amino acid at position 1 (glutamic acid) has been changed to aspartic acid.

In one aspect, the invention provides the following polypeptide:
a) SEQ ID NO: 142 to SEQ ID NO: 145 and SEQ ID NO: 162 to SEQ ID NO: 165;
b) a polypeptide having an amino acid difference of no more than 3, preferably no more than 2, and more preferably no more than one of SEQ ID NOs: 142 to 145 and SEQ ID NOs: 162 to 165,
i) the first amino acid sequence or Nanobody® included in the polypeptide has aspartic acid (Asp, D) at position 1;
ii) the polypeptide has the same, about the same, or a higher affinity as compared to a polypeptide that does not have three, two or one amino acid difference (as the affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or wherein said polypeptide has the same, about the same, or higher potency (as compared to a polypeptide without three, two or one amino acid difference) (As defined herein) is provided.

  A preferred trivalent polypeptide of the invention comprises or consists essentially of SEQ ID NO: 142. Another preferred trivalent polypeptide of the invention comprises or consists essentially of SEQ ID NO: 143. Another preferred trivalent polypeptide of the invention comprises or consists essentially of SEQ ID NO: 144. Another preferred trivalent polypeptide of the invention comprises or consists essentially of SEQ ID NO: 145. Another preferred trivalent polypeptide of the invention comprises or consists essentially of one of SEQ ID NOs: 162-165.

  In another preferred aspect, the invention provides a trivalent polypeptide comprising, or consisting essentially of, SEQ ID NO: 53, wherein the glutamic acid at position 1 has been changed to aspartic acid (Glu1Asp).

  In another preferred aspect, the invention provides a trivalent polypeptide comprising, or consisting essentially of, SEQ ID NO: 77, wherein the glutamic acid at position 1 has been changed to aspartic acid (Glu1Asp).

  In another preferred aspect, the invention provides a trivalent polypeptide comprising, or consisting essentially of, SEQ ID NO: 78, wherein glutamic acid at position 1 has been changed to aspartic acid (Glu1Asp).

  In another preferred aspect, the invention provides a trivalent polypeptide comprising, or consisting essentially of, SEQ ID NO: 79, wherein glutamic acid at position 1 has been changed to aspartic acid (Glu1Asp).

  In another preferred aspect, the invention provides a trivalent polypeptide comprising, or consisting essentially of, SEQ ID NO: 158, wherein the glutamic acid at position 1 has been changed to aspartic acid (Glu1Asp).

  In another preferred aspect, the invention provides a trivalent polypeptide comprising, or consisting essentially of, SEQ ID NO: 159, wherein the glutamic acid at position 1 has been changed to aspartic acid (Glu1Asp).

  In another preferred aspect, the invention provides a trivalent polypeptide comprising, or consisting essentially of, SEQ ID NO: 160, wherein the glutamic acid at position 1 has been changed to aspartic acid (Glu1Asp).

  In another preferred aspect, the invention provides a trivalent polypeptide comprising, or consisting essentially of, SEQ ID NO: 161, wherein the glutamic acid at position 1 has been changed to aspartic acid (Glu1Asp).

  In another preferred embodiment, the invention relates to at least one (preferably two, more preferably all three) Nanobodies® forming part of SEQ ID NO: 53, wherein one or more (eg 2 , 3, 4, 5, 6, 7, 8, or 9, 10, 11, or 12) amino acid residues are Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser , Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp, wherein the glutamic acid at position 1 has been changed to aspartic acid (Glu1Asp), comprising or consisting essentially of SEQ ID NO: 53. A trivalent polypeptide is provided.

  In another preferred embodiment, the invention relates to at least one (preferably two, more preferably all three) Nanobodies® forming part of SEQ ID NO: 53, wherein one or more (eg 2 , 3, 4, 5, 6, 7, 8, or 9) amino acid residues are selected from Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp. A trivalent polypeptide comprising, or consisting essentially of, SEQ ID NO: 53, wherein the glutamic acid at position 1 has been changed to aspartic acid (Glu1Asp).

  In another preferred embodiment, the invention relates to at least one (preferably two, more preferably all three) Nanobodies® forming part of SEQ ID NO: 53, wherein one or more (eg 2 , Three or four) amino acid residues have undergone a mutation selected from Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu, and the glutamic acid at position 1 has been changed to aspartic acid (Glu1Asp). A trivalent polypeptide comprising or consisting essentially of No. 53 is provided.

  In another preferred embodiment, the invention relates to at least one (preferably two, more preferably all three) Nanobodies® forming part of SEQ ID NO: 53, wherein one or more (eg 2 , 3, 4, 5, 6, or 7) amino acid residues have undergone a mutation selected from Ala14Pro, Ser19Arg, Ile20Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu, and the first position Glutamic acid has been changed to aspartic acid (Glu1Asp), comprising or consisting essentially of SEQ ID NO: 53.

In another preferred embodiment, the invention relates to at least one (preferably two, more preferably all three) Nanobodies® forming part of SEQ ID NO: 53, wherein the amino acid residue is:
Glu1Asp,
Glu1Asp, Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Glu1Asp, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Glu1Asp, Gly78Leu, Ala83Arg, Asp85Glu, and Arg105Gln;
Glu1Asp, Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Glu1Asp, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Glu1Asp, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54Asp,
Glu1Asp and Gly54Asp,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Ala83Arg, Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Asp85Glu,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Arg105Gln,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Asp85Glu,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Arg105Gln,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Asp85Glu, and Arg105Gln, or Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Ag, ArA85Ag, ArA85Ag, ArA85Ag
A trivalent polypeptide comprising or consisting essentially of SEQ ID NO: 53, mutated to

  In another preferred aspect, a polypeptide of the invention comprises or consists essentially of the amino acid sequence of SEQ ID NO: 142. In another preferred embodiment, a polypeptide of the invention comprises or consists essentially of the amino acid sequence of SEQ ID NO: 143. In another preferred embodiment, a polypeptide of the invention comprises or consists essentially of the amino acid sequence of SEQ ID NO: 144. In another preferred aspect, a polypeptide of the invention comprises or consists essentially of the amino acid sequence of SEQ ID NO: 145. In another preferred aspect, a polypeptide of the invention comprises or consists essentially of one of SEQ ID NOs: 162-165.

Polypeptide comprising the amino acid sequence and polypeptide sequence as described above, for example, improved stability, reduced immunogenicity, (as further described herein (actual or apparent) K _D value, ( actual or apparent) _{K a value k on} rate and / or _{k off} rate, or alternatively is preferably measured as _{IC 50} values, and / or represented by) improved binding properties, for the protein F of hRSV For use as a prophylactically, therapeutically and / or pharmacologically active factor such as improved affinity and / or improved binding activity and / or improved efficacy and / or efficacy to neutralize hRSV. Demonstrated beneficial properties.

More specifically, these polypeptides and amino acid sequences of the present invention preferably have a dissociation constant (K) of 100 nM to 0.1 nM or less, preferably 10 nM to 0.1 nM or less, more preferably 1 nM to 0.1 nM or less. _D ) such that it binds to protein F of hRSV and / or from 10 ⁴ M ⁻¹ s ⁻¹ to about 10 ⁷ M ⁻¹ s ⁻¹ , preferably 10 ⁵ M ⁻¹ s ⁻¹ to 10 ⁷ M ^−. Binding to protein F of hRSV at a k _on rate of ¹ s ⁻¹ , more preferably about 10 ⁶ M ⁻¹ s ⁻¹ or more, and / or 10 ⁻² s ⁻¹ (t _1/2 = 0. 69 s) to 10 ^-4 s ^-1 (in the case of a substantially irreversible complex where t _1/2 is several days), preferably 10 ^-3 s ^-1 to 10 ^-4 s ^-1 , more preferably 5 × 10 ^-3 s ^-1 ~10 ^-4 s ¹ in the following _{k off} rate to bind to protein F of hRSV,
(As further described herein (actual or apparent) _{K D} value, (actual or apparent) _{K A value k on} rate and / or _{k off} rate, or alternatively suitable as an _{IC 50} value Can bind to protein F of hRSV with an affinity (measured and / or expressed).

  Some preferred IC50 values for the binding of the polypeptides of the invention to protein F of hRSV will become clear from the further description and examples herein.

  Assays for determining the IC50 include competition assays such as competition ELISAs (eg, competition with Synagis® or Fab fragments thereof), or more preferably Anderson et al. (1985, J. Clin. Microbiol. 22: 1050). -1052), a neutralization assay such as a variation of this assay as described in Example 6, or Johnson et al. (1997, J. Inf. Dis. 176: 1215-1224). ) Include plaque reduction assays as described, and variations thereof.

  For example, in a competition assay with SYNAGIS®, a polypeptide of the invention may have an IC50 value of 1 nM to 100 nM, preferably 10 nM to 50 nM or less.

  For example, in a neutralization assay of a RSV Long strain (e.g., as described in Example 6), the polypeptide of the invention has an IC50 value of 10 pM to 1000 pM, preferably 10 pM to 250 pM, more preferably 50 pM to 200 pM or less. Can have. In a neutralization assay, the polypeptides of the invention have at least the same, preferably better, at least 10 times better, preferably 20 times better compared to the IC50 values obtained with SYNAGIS®. , More preferably 50 times better, even more preferably 60 times, 70 times, 80 times or more than 80 times better.

  The present invention relates to monovalent polypeptides or constructs comprising (or consisting essentially of) one amino acid sequence or Nanobody (R) of the invention ("monovalent polypeptide of the invention" or " Also referred to as "monovalent constructs"). Preferred monovalent constructs of the invention comprise one of SEQ ID NOs: 60-76, such as SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76. One, for example, SEQ ID NO: 65 or SEQ ID NO: 75, one of SEQ ID NO: 138 to SEQ ID NO: 141, one of SEQ ID NO: 146 to SEQ ID NO: 153, or one of SEQ ID NO: 154 to SEQ ID NO: 157 Or essentially consists of one. Such monovalent constructs, as well as the amino acid sequences and Nanobodies® of the invention, can be used in the preparation of a polypeptide of the invention, for example, a polyvalent polypeptide of the invention as described above.

  The polypeptides of the present invention generally comprise an amino acid sequence, Nanobody® or monovalent construct of the invention, optionally via one or more suitable linkers, and one or more additional amino acids of the invention. It can be prepared by a method comprising at least the step of suitably linking a sequence, Nanobody® or a monovalent construct to provide a polypeptide of the present invention. The polypeptide of the present invention generally includes at least a step of preparing a nucleic acid encoding the polypeptide of the present invention, a step of expressing the nucleic acid by a suitable method, and a step of collecting the expressed polypeptide of the present invention. It can also be prepared by a method. Such methods can be carried out in a manner known per se, which will be clear to the skilled person, for example based on the methods and techniques further described herein.

  The method of preparing multivalent, multiparatopic and / or multispecific amino acids or polypeptides of the invention comprises combining two or more monovalent amino acid sequences or monovalent constructs of the invention with, for example, one or more It may include at least the step of linking the linker and the linker together in a suitable manner. Monovalent constructs (and linkers) are known in the art and can be attached by any method as further described herein. Preferred techniques include ligation of nucleic acid sequences encoding monovalent constructs (and linkers) to prepare genetic constructs that express multivalent, multiparatopic and / or multispecific amino acids or polypeptides. Techniques for linking amino acid or nucleic acid sequences will be apparent to those of skill in the art, and refer to the standard handbooks described above (eg, Sambrook et al. And Ausubel et al.), And the Examples below.

  Accordingly, the present invention also relates to the use of an amino acid sequence, Nanobody® or monovalent construct of the invention in preparing a multivalent polypeptide of the invention. The method of preparing a multivalent polypeptide comprises the steps of: providing an amino acid sequence, Nanobody® or monovalent construct of the invention, optionally via one or more linkers, and at least one additional amino acid sequence of the invention, Nanobody ® or monovalent constructs. Thus, the amino acid sequence, Nanobody®, or monovalent construct can bind two (eg, in a divalent polypeptide), three (eg, in a trivalent polypeptide) or more (eg, in a multivalent polypeptide). Used as a binding domain or unit in providing and / or preparing a multivalent polypeptide comprising the unit. In this regard, the amino acid sequences, Nanobodies® and monovalent constructs provide a polyvalent, preferably divalent or trivalent, polypeptide of the invention comprising two, three or more binding units and And / or can be used as a binding domain or unit during preparation. Preferably, the binding domains or units are linked via a linker such that the multivalent polypeptide preferably exhibits intramolecular bonds as compared to intermolecular bonds. Also preferably, the multivalent polypeptide is capable of binding simultaneously to both or all three binding sites on the RSV F protein.

  Accordingly, the present invention also relates to the use of the amino acid sequences or Nanobodies® of the present invention (as described above) in preparing multivalent polypeptides. The method of preparing a multivalent polypeptide comprises the steps of optionally providing, via one or more linkers, an amino acid sequence or Nanobody® of the invention and at least one further amino acid sequence or Nanobody® of the invention. Including concatenation with

In a preferred embodiment, the present invention provides a method for preparing a multivalent polypeptide comprising the steps of: a) SEQ ID NO: 60 to SEQ ID NO: 76;
b) an amino acid sequence having only one amino acid difference from one of SEQ ID NO: 60 to SEQ ID NO: 76, preferably only two, more preferably only one;
i) the amino acid sequence has glutamine (Gln, Q) at position 105 (the position is determined by Kabat numbering);
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) (As defined herein) relates to the use of an amino acid sequence selected from amino acid sequences. A method for preparing a multivalent polypeptide comprises linking the above amino acid sequence to at least one additional amino acid sequence, optionally via one or more linkers.

  In a preferred embodiment, the amino acid sequence used in preparing the multivalent polypeptide comprises or consists essentially of one of SEQ ID NO: 60-SEQ ID NO: 76.

In another preferred aspect, the invention provides a method for preparing a multivalent polypeptide comprising the steps of: a) SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76;
b) only three, preferably only two and more preferably only one amino acid difference from one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75 and SEQ ID NO: 76 An amino acid sequence having
i) the amino acid sequence has glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and / or arginine (Arg, R) at position 83 (the above positions are determined by Kabat numbering; Ii) the amino acid sequence has the same, substantially the same or higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (the affinity is measured by surface plasmon resonance) That binds to protein F of hRSV and / or wherein said amino acid sequence is the same, approximately the same, or as compared to an amino acid sequence that has no three, two or one amino acid difference. It relates to the use of an amino acid sequence selected from the amino acid sequences, which shall have a higher potency (as defined herein). A method for preparing a multivalent polypeptide comprises linking the above amino acid sequence to at least one additional amino acid sequence, optionally via one or more linkers.

In another preferred aspect, the invention provides a method for preparing a multivalent polypeptide comprising the steps of: a) SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76; SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76 with only 3, preferably 2, and more preferably only 1 amino acid difference from one And
i) the amino acid sequence has glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and arginine (Arg, R) at position 83 (the above positions are determined by Kabat numbering) ),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) (As defined herein) relates to the use of an amino acid sequence selected from amino acid sequences. A method for preparing a multivalent polypeptide comprises linking the above amino acid sequence to at least one additional amino acid sequence, optionally via one or more linkers.

  In a preferred embodiment, the amino acid sequence used in preparing the multivalent polypeptide comprises one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76 Or consisting essentially of:

In another preferred aspect, the invention provides a method for preparing a multivalent polypeptide comprising the steps of: a) SEQ ID NO: 65 and SEQ ID NO: 76;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NO: 65 and SEQ ID NO: 76,
i) the amino acid sequence is aspartic acid (Asp, D) at position 54, glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and / or arginine (Arg, R) at position 83; (The position is determined by Kabat numbering),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) (As defined herein) relates to the use of an amino acid sequence selected from amino acid sequences. A method for preparing a multivalent polypeptide comprises linking the above amino acid sequence to at least one additional amino acid sequence, optionally via one or more linkers.

In another preferred aspect, the invention provides a method for preparing a multivalent polypeptide comprising the steps of: a) SEQ ID NO: 65 and SEQ ID NO: 76;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NO: 65 and SEQ ID NO: 76,
i) the amino acid sequence has aspartic acid (Asp, D) at position 54, glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and arginine (Arg, R) at position 83; (The above position is obtained by Kabat numbering)
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) (As defined herein) relates to the use of an amino acid sequence selected from amino acid sequences. A method for preparing a multivalent polypeptide comprises linking the above amino acid sequence to at least one additional amino acid sequence, optionally via one or more linkers.

  In a preferred embodiment, the amino acid sequence used in preparing the multivalent polypeptide comprises or consists essentially of SEQ ID NO: 65. In another preferred embodiment, the amino acid sequence used in preparing the multivalent polypeptide comprises or consists essentially of SEQ ID NO: 76.

In a preferred embodiment, the present invention provides a method for preparing a multivalent polypeptide comprising the steps of: a) SEQ ID NO: 146 to SEQ ID NO: 153;
b) an amino acid sequence having only one, preferably two, more preferably only one amino acid difference from one of SEQ ID NOs: 146 to 153,
i) the amino acid sequence comprises proline (Pro, P) at position 14, arginine (Arg, R) at position 19, leucine (Leu, L) at position 20 and / or leucine (Leu, L) at position 108 Have (the above positions are determined by Kabat numbering),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) (As defined herein) relates to the use of an amino acid sequence selected from amino acid sequences. A method for preparing a multivalent polypeptide comprises linking the above amino acid sequence to at least one additional amino acid sequence, optionally via one or more linkers.

  In a preferred embodiment, the amino acid sequence used in preparing the multivalent polypeptide comprises or consists essentially of one of SEQ ID NOs: 146-153.

In another preferred aspect, the invention provides a method for preparing a multivalent polypeptide comprising the steps of: a) SEQ ID NOs: 146-149 and SEQ ID NOs: 151-153;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153,
i) the amino acid sequence comprises proline (Pro, P) at position 14, arginine (Arg, R) at position 19, leucine (Leu, L) at position 20, and / or leucine (Leu, L) at position 108 Further having arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85, and / or glutamine (Gln, Q) at position 105 (the position is determined by Kabat numbering),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) (As defined herein) relates to the use of an amino acid sequence selected from amino acid sequences. A method for preparing a multivalent polypeptide comprises linking the above amino acid sequence to at least one additional amino acid sequence, optionally via one or more linkers.

In another preferred aspect, the invention provides a method for preparing a multivalent polypeptide comprising the steps of: a) SEQ ID NOs: 146-149 and SEQ ID NOs: 151-153;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153,
i) the amino acid sequence is
SEQ ID NO: 146 (this amino acid sequence preferably has glutamine (Gln, Q) at position 105);
SEQ ID NO: 147 (this amino acid sequence preferably has arginine (Arg, R) at position 83 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 148 (this amino acid sequence preferably has glutamic acid (Glu, E) at position 85 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 149 (this amino acid sequence preferably has arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 151 (this amino acid sequence preferably has arginine (Arg, R) at position 83);
SEQ ID NO: 152 (this amino acid sequence preferably has glutamic acid (Glu, E) at position 85);
SEQ ID NO: 153 (this amino acid sequence preferably has arginine (Arg, R) at position 83 and glutamic acid (Glu, E) at position 85)
The amino acid sequence has a proline (Pro, P) at position 14 and an arginine (Arg, R) at position 19, as in the case of having only 3, preferably 2, and more preferably only 1 amino acid difference. Leucine (Leu, L) at position 20 and / or leucine (Leu, L) at position 108, arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85, and / or position 105 Has glutamine (Gln, Q) (the position is determined by Kabat numbering),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) (As defined herein) relates to the use of an amino acid sequence selected from amino acid sequences. A method for preparing a multivalent polypeptide comprises linking the above amino acid sequence to at least one additional amino acid sequence, optionally via one or more linkers.

    In a preferred embodiment, the amino acid sequence used in preparing the multivalent polypeptide comprises or consists essentially of one of SEQ ID NOs: 146-149 and SEQ ID NOs: 151-153.

  In another preferred aspect, the invention provides a method for preparing one or more (eg, 2, 3, 4, 5, 6, 7, 8, or 9, (10, 11, or 12) amino acid residues were mutated from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54A selected from Gly54A. It relates to the use of an amino acid sequence comprising or consisting essentially of SEQ ID NO: 5.

  In another preferred aspect, the invention provides one or more (eg, 2, 3, 4, 5, 6, 7, 8, or 9) in preparing a multivalent polypeptide. Comprises or consists essentially of SEQ ID NO: 5, wherein the amino acid residue of SEQ ID NO: 5 has a mutation selected from Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp. About use.

  In another preferred embodiment, the present invention provides that the method comprises preparing one or more (eg 2, 3 or 4) amino acid residues from Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu in preparing a multivalent polypeptide. The invention relates to the use of an amino acid sequence comprising or consisting essentially of SEQ ID NO: 5, having a selected mutation.

  In another preferred embodiment, the present invention provides a method for preparing a multivalent polypeptide, wherein one or more (eg, 2, 3, 4, 5, 6, or 7) amino acid residues are The present invention relates to the use of an amino acid sequence comprising or consisting essentially of SEQ ID NO: 5, which has been mutated selected from Ala14Pro, Ser19Arg, Ile20Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu.

In another preferred embodiment, the present invention provides a method for preparing a multivalent polypeptide, wherein the amino acid residue is:
Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Gly78Leu, Ala83Arg, Asp85Glu, and Arg105Gln,
Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54Asp,
Gly54Asp,
Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Ala83Arg,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Asp85Glu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Arg105Gln,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Asp85Glu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Arg105Gln,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Asp85Glu, and Arg105Gln, or Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, Asp85Glu, and Arg105Gln.
The use of an amino acid sequence comprising or consisting essentially of SEQ ID NO: 5 mutated.

  In another preferred embodiment, the present invention relates to the use of an amino acid sequence selected from the above, wherein the amino acid at position 1 (glutamic acid) is changed to aspartic acid in preparing a multivalent polypeptide.

In this regard, the present invention provides a method for preparing a multivalent polypeptide comprising the steps of: a) SEQ ID NO: 138 to SEQ ID NO: 141 and SEQ ID NO: 154 to SEQ ID NO: 157;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 138 to 141 and 154 to 157,
i) the amino acid sequence has aspartic acid (Asp, D) at position 1 (the position is determined by Kabat numbering);
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) (As defined herein) relates to the use of an amino acid sequence selected from amino acid sequences. A method for preparing a multivalent polypeptide comprises linking the above amino acid sequence to at least one additional amino acid sequence, optionally via one or more linkers.

  In a preferred embodiment, the amino acid sequence used in preparing the multivalent polypeptide comprises or consists essentially of SEQ ID NO: 138. In another preferred embodiment, the amino acid sequence used in preparing the multivalent polypeptide comprises or consists essentially of SEQ ID NO: 139. In another preferred embodiment, the amino acid sequence used in preparing the multivalent polypeptide comprises or consists essentially of SEQ ID NO: 140. In another preferred embodiment, the amino acid sequence used in preparing the multivalent polypeptide comprises or consists essentially of SEQ ID NO: 141. In another preferred embodiment, the amino acid sequence used in preparing the multivalent polypeptide comprises or consists essentially of one of SEQ ID NOs: 154-157.

  In another preferred aspect, the invention provides the use of an amino acid sequence comprising or consisting essentially of SEQ ID NO: 5, wherein glutamic acid at position 1 has been changed to aspartic acid in preparing a multivalent polypeptide. About.

  In another preferred aspect, the invention provides the use of an amino acid sequence comprising or consisting essentially of SEQ ID NO: 62 wherein glutamic acid at position 1 has been changed to aspartic acid in preparing a multivalent polypeptide. About.

  In another preferred aspect, the invention provides the use of an amino acid sequence comprising or consisting essentially of SEQ ID NO: 65, wherein glutamic acid at position 1 has been changed to aspartic acid in preparing a multivalent polypeptide. About.

  In another preferred aspect, the invention provides the use of an amino acid sequence comprising or consisting essentially of SEQ ID NO: 76, wherein glutamic acid at position 1 has been changed to aspartic acid in the preparation of a multivalent polypeptide. About.

  In another preferred aspect, the invention provides the use of an amino acid sequence comprising or consisting essentially of SEQ ID NO: 75, wherein glutamic acid at position 1 has been changed to aspartic acid in preparing a multivalent polypeptide. About.

  In another preferred aspect, the invention provides the use of an amino acid sequence comprising or consisting essentially of SEQ ID NO: 147, wherein glutamic acid at position 1 has been changed to aspartic acid in the preparation of a multivalent polypeptide. About.

  In another preferred aspect, the invention provides the use of an amino acid sequence comprising or consisting essentially of SEQ ID NO: 149, wherein glutamic acid at position 1 has been changed to aspartic acid in the preparation of a multivalent polypeptide. About.

  In another preferred aspect, the invention provides the use of an amino acid sequence comprising or consisting essentially of SEQ ID NO: 153, wherein glutamic acid at position 1 has been changed to aspartic acid in preparing a multivalent polypeptide. About.

  In another preferred aspect, the invention provides a method for preparing one or more (eg, 2, 3, 4, 5, 6, 7, 8, or 9, (10, 11, or 12) amino acid residues are mutated from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and GlyA from Gly54A. 1, wherein the glutamic acid at position 1 has been changed to aspartic acid, comprising or consisting essentially of SEQ ID NO: 5.

  In another preferred aspect, the invention provides one or more (eg, 2, 3, 4, 5, 6, 7, 8, or 9) in preparing a multivalent polypeptide. Has undergone a mutation selected from Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp, and the glutamic acid at position 1 has been changed to aspartic acid. 5. Use of an amino acid sequence comprising or consisting essentially of 5.

  In another preferred embodiment, the present invention provides that the method comprises preparing one or more (eg 2, 3 or 4) amino acid residues from Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu in preparing a multivalent polypeptide. The present invention relates to the use of an amino acid sequence comprising or consisting essentially of SEQ ID NO: 5, which has undergone a selected mutation and wherein the glutamic acid at position 1 has been changed to aspartic acid.

  In another preferred embodiment, the present invention provides a method for preparing a multivalent polypeptide, wherein one or more (eg, 2, 3, 4, 5, 6, or 7) amino acid residues are A mutation comprising a mutation selected from Ala14Pro, Ser19Arg, Ile20Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu, wherein the glutamic acid at position 1 has been changed to aspartic acid, comprising or essentially consisting of SEQ ID NO: 5. The use of an amino acid sequence comprising:

In another preferred embodiment, the present invention provides a method for preparing a multivalent polypeptide, wherein the amino acid residue is:
Glu1Asp,
Glu1Asp, Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Glu1Asp, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Glu1Asp, Gly78Leu, Ala83Arg, Asp85Glu, and Arg105Gln;
Glu1Asp, Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Glu1Asp, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Glu1Asp, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54Asp,
Glu1Asp and Gly54Asp,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Ala83Arg;
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Asp85Glu,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Arg105Gln,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Asp85Glu,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Arg105Gln,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Asp85Glu, and Arg105Gln, or Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Ag, ArA85Ag, ArA85Ag, ArA85Ag
The use of an amino acid sequence comprising or consisting essentially of SEQ ID NO: 5 mutated.

  The present invention also relates to the use of two inventive amino acid sequences and / or Nanobodies® (as described above) in preparing a bivalent polypeptide. The method of preparing a bivalent polypeptide comprises linking the amino acid sequence and / or Nanobody® of the invention, optionally via a linker.

Accordingly, in a preferred embodiment, the present invention provides a method for preparing a bivalent polypeptide comprising the steps of: a) SEQ ID NO: 60 to SEQ ID NO: 76;
b) an amino acid sequence having only one amino acid difference from one of SEQ ID NO: 60 to SEQ ID NO: 76, preferably only two, more preferably only one;
i) the amino acid sequence has glutamine (Gln, Q) at position 105 (the position is determined by Kabat numbering);
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) (As defined herein) relates to the use of two (preferably identical) amino acid sequences selected from amino acid sequences. Methods for preparing bivalent polypeptides include the ligation of each of the above amino acid sequences, optionally via a linker.

  In a preferred embodiment, the two amino acid sequences used in preparing the bivalent polypeptide comprise or consist essentially of one of SEQ ID NO: 60-SEQ ID NO: 76.

In another preferred aspect, the invention provides a method of preparing a bivalent polypeptide comprising the steps of: a) SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76;
b) only three, preferably only two and more preferably only one amino acid difference from one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75 and SEQ ID NO: 76 An amino acid sequence having
i) the amino acid sequence has glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and / or arginine (Arg, R) at position 83 (the above positions are determined by Kabat numbering; Desired),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) (As defined herein) relates to the use of two (preferably identical) amino acid sequences selected from amino acid sequences. The method of preparing a bivalent polypeptide comprises linking the above amino acid sequences, optionally via a linker.

In another preferred aspect, the invention provides a method of preparing a bivalent polypeptide comprising the steps of: a) SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76;
b) only three, preferably only two and more preferably only one amino acid difference from one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75 and SEQ ID NO: 76 An amino acid sequence having
i) the amino acid sequence has glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and arginine (Arg, R) at position 83 (the above positions are determined by Kabat numbering) ),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) (As defined herein) relates to the use of two (preferably identical) amino acid sequences selected from amino acid sequences. The method of preparing a bivalent polypeptide comprises linking the above amino acid sequences, optionally via a linker.

  In a preferred embodiment, the two amino acid sequences used in preparing the bivalent polypeptide are one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76. Or consists essentially of.

In another preferred aspect, the invention provides a method for preparing a bivalent polypeptide comprising the steps of: a) SEQ ID NO: 65 and SEQ ID NO: 76;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NO: 65 and SEQ ID NO: 76,
i) the amino acid sequence is aspartic acid (Asp, D) at position 54, glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and / or arginine (Arg, R) at position 83; (The position is determined by Kabat numbering),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) (As defined herein) relates to the use of two (preferably identical) amino acid sequences selected from amino acid sequences. The method of preparing a bivalent polypeptide comprises linking the above amino acid sequences, optionally via a linker.

In another preferred aspect, the invention provides a method for preparing a bivalent polypeptide comprising the steps of: a) SEQ ID NO: 65 and SEQ ID NO: 76;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NO: 65 and SEQ ID NO: 76,
i) the amino acid sequence has aspartic acid (Asp, D) at position 54, glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and arginine (Arg, R) at position 83; (The above position is obtained by Kabat numbering)
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) (As defined herein) relates to the use of two (preferably identical) amino acid sequences selected from amino acid sequences. The method of preparing a bivalent polypeptide comprises linking the above amino acid sequences, optionally via a linker.

In another preferred embodiment, the present invention provides a method for preparing a bivalent polypeptide comprising the steps of: a) SEQ ID NO: 146 to SEQ ID NO: 153;
b) an amino acid sequence having only one, preferably two, more preferably only one amino acid difference from one of SEQ ID NOs: 146 to 153,
i) the amino acid sequence comprises proline (Pro, P) at position 14, arginine (Arg, R) at position 19, leucine (Leu, L) at position 20 and / or leucine (Leu, L) at position 108 Have (the above positions are determined by Kabat numbering),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) (As defined herein) relates to the use of two (preferably identical) amino acid sequences selected from amino acid sequences. The method of preparing a bivalent polypeptide comprises linking the above amino acid sequence to at least one further amino acid sequence, optionally via one or more linkers.

  In a preferred embodiment, the amino acid sequence used in preparing the bivalent polypeptide comprises or consists essentially of one of SEQ ID NOs: 146-153.

In another preferred aspect, the present invention provides a method for preparing a bivalent polypeptide comprising the steps of: a) SEQ ID NOs: 146-149 and SEQ ID NOs: 151-153;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153,
i) the amino acid sequence comprises proline (Pro, P) at position 14, arginine (Arg, R) at position 19, leucine (Leu, L) at position 20, and / or leucine (Leu, L) at position 108 Further having arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85, and / or glutamine (Gln, Q) at position 105 (the position is determined by Kabat numbering),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) (As defined herein) relates to the use of two (preferably identical) amino acid sequences selected from amino acid sequences. The method of preparing a bivalent polypeptide comprises linking the above amino acid sequence to at least one further amino acid sequence, optionally via one or more linkers.

In another preferred aspect, the present invention provides a method for preparing a bivalent polypeptide comprising the steps of: a) SEQ ID NOs: 146-149 and SEQ ID NOs: 151-153;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153,
i) the amino acid sequence is
SEQ ID NO: 146 (this amino acid sequence preferably has glutamine (Gln, Q) at position 105);
SEQ ID NO: 147 (this amino acid sequence preferably has arginine (Arg, R) at position 83 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 148 (this amino acid sequence preferably has glutamic acid (Glu, E) at position 85 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 149 (this amino acid sequence preferably has arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 151 (this amino acid sequence preferably has arginine (Arg, R) at position 83);
SEQ ID NO: 152 (this amino acid sequence preferably has glutamic acid (Glu, E) at position 85);
SEQ ID NO: 153 (this amino acid sequence preferably has arginine (Arg, R) at position 83 and glutamic acid (Glu, E) at position 85)
The amino acid sequence has a proline (Pro, P) at position 14 and an arginine (Arg, R) at position 19, as in the case of having only 3, preferably 2, and more preferably only 1 amino acid difference. Leucine (Leu, L) at position 20 and / or leucine (Leu, L) at position 108, arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85, and / or position 105 Has glutamine (Gln, Q) (the position is determined by Kabat numbering),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) (As defined herein) relates to the use of two (preferably identical) amino acid sequences selected from amino acid sequences. The method of preparing a bivalent polypeptide comprises linking the above amino acid sequence to at least one further amino acid sequence, optionally via one or more linkers.

  In a preferred embodiment, the amino acid sequence used in preparing the bivalent polypeptide comprises or consists essentially of one of SEQ ID NOs: 146-149 and SEQ ID NOs: 151-153.

  In another preferred aspect, the invention provides a method for preparing one or more (eg, 2, 3, 4, 5, 6, 7, 8, or 9, (10, 11, or 12) amino acid residues were mutated from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54A selected from Gly54A. It relates to the use of two (preferably identical) amino acid sequences comprising or consisting essentially of SEQ ID NO: 5.

  In another preferred aspect, the invention provides one or more (eg, 2, 3, 4, 5, 6, 7, 8, or 9) in preparing a bivalent polypeptide. Amino acid residues comprising or consisting essentially of SEQ ID NO: 5, having a mutation selected from Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp. (Preferably identical) amino acid sequences.

  In another preferred embodiment, the present invention provides that the method of preparing a bivalent polypeptide comprises the step of preparing one or more (eg 2, 3, or 4) amino acid residues from Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu. The present invention relates to the use of two (preferably identical) amino acid sequences comprising or consisting essentially of SEQ ID NO: 5, having undergone a selected mutation.

  In another preferred embodiment, the present invention provides a method for preparing a bivalent polypeptide, wherein one or more (eg, 2, 3, 4, 5, 6, or 7) amino acid residues are For the use of two (preferably identical) amino acid sequences comprising or consisting essentially of SEQ ID NO: 5, mutated selected from Ala14Pro, Ser19Arg, Ile20Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu. .

In another preferred embodiment, the present invention provides a method for preparing a bivalent polypeptide, wherein the amino acid residue is
Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Gly78Leu, Ala83Arg, Asp85Glu, and Arg105Gln,
Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54Asp, or Gly54Asp,
Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Ala83Arg,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Asp85Glu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Arg105Gln,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Asp85Glu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Arg105Gln,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Asp85Glu, and Arg105Gln, or Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, Asp85Glu, and Arg105Gln.
The use of two (preferably identical) amino acid sequences comprising or consisting essentially of SEQ ID NO: 5, having been mutated.

  In another preferred embodiment, the present invention relates to the use of an amino acid sequence selected from the above, wherein the amino acid at position 1 (glutamic acid) has been changed to aspartic acid in the preparation of a bivalent polypeptide.

In this regard, the present invention provides a method for preparing a bivalent polypeptide comprising the steps of: a) SEQ ID NOs: 138-141 and SEQ ID NOs: 154-157;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 138 to 141 and 154 to 157,
i) the amino acid sequence has aspartic acid (Asp, D) at position 1 (the position is determined by Kabat numbering);
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) (As defined herein) relates to the use of an amino acid sequence selected from amino acid sequences. The method of preparing a bivalent polypeptide comprises linking the above amino acid sequence to at least one further amino acid sequence, optionally via one or more linkers.

  In a preferred embodiment, the amino acid sequence used in preparing the bivalent polypeptide comprises or consists essentially of SEQ ID NO: 138. In another preferred embodiment, the amino acid sequence used in preparing the bivalent polypeptide comprises or consists essentially of SEQ ID NO: 139. In another preferred embodiment, the amino acid sequence used in preparing the bivalent polypeptide comprises or consists essentially of SEQ ID NO: 140. In another preferred embodiment, the amino acid sequence used in preparing the bivalent polypeptide comprises or consists essentially of SEQ ID NO: 141. In another preferred embodiment, the amino acid sequence used in preparing the bivalent polypeptide comprises or consists essentially of SEQ ID NO: 154-SEQ ID NO: 157.

  In another preferred aspect, the invention provides the use of an amino acid sequence comprising or consisting essentially of SEQ ID NO: 5, wherein glutamic acid at position 1 has been changed to aspartic acid in the preparation of a bivalent polypeptide. About.

  In another preferred aspect, the invention provides the use of an amino acid sequence comprising or consisting essentially of SEQ ID NO: 62 wherein glutamic acid at position 1 has been changed to aspartic acid in preparing a bivalent polypeptide. About.

  In another preferred aspect, the invention provides for the use of an amino acid sequence comprising or consisting essentially of SEQ ID NO: 65, wherein glutamic acid at position 1 has been changed to aspartic acid in the preparation of a bivalent polypeptide. About.

  In another preferred aspect, the invention provides for the use of an amino acid sequence comprising or consisting essentially of SEQ ID NO: 76, wherein glutamic acid at position 1 has been changed to aspartic acid in the preparation of a bivalent polypeptide. About.

  In another preferred aspect, the invention provides the use of an amino acid sequence comprising or consisting essentially of SEQ ID NO: 75, wherein glutamic acid at position 1 has been changed to aspartic acid in the preparation of a bivalent polypeptide. About.

  In another preferred aspect, the invention provides the use of an amino acid sequence comprising or consisting essentially of SEQ ID NO: 147, wherein glutamic acid at position 1 has been changed to aspartic acid in the preparation of a bivalent polypeptide. About.

  In another preferred aspect, the invention provides for the use of an amino acid sequence comprising or consisting essentially of SEQ ID NO: 149, wherein glutamic acid at position 1 has been changed to aspartic acid in the preparation of a bivalent polypeptide. About.

  In another preferred aspect, the invention provides the use of an amino acid sequence comprising or consisting essentially of SEQ ID NO: 153, wherein glutamic acid at position 1 has been changed to aspartic acid in preparing a bivalent polypeptide. About.

  In another preferred aspect, the invention provides a method for preparing one or more (eg, 2, 3, 4, 5, 6, 7, 8, or 9, (10, 11, or 12) amino acid residues are mutated from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and GlyA from Gly54A. 1, wherein the glutamic acid at position 1 has been changed to aspartic acid, comprising or consisting essentially of SEQ ID NO: 5.

  In another preferred aspect, the invention provides one or more (eg, 2, 3, 4, 5, 6, 7, 8, or 9) in preparing a bivalent polypeptide. Has undergone a mutation selected from Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp, and the glutamic acid at position 1 has been changed to aspartic acid. 5. Use of an amino acid sequence comprising or consisting essentially of 5.

  In another preferred embodiment, the present invention provides that the method of preparing a bivalent polypeptide comprises the step of preparing one or more (eg 2, 3, or 4) amino acid residues from Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu. The present invention relates to the use of an amino acid sequence comprising or consisting essentially of SEQ ID NO: 5, which has undergone a selected mutation and wherein the glutamic acid at position 1 has been changed to aspartic acid.

  In another preferred embodiment, the present invention provides a method for preparing a bivalent polypeptide, wherein one or more (eg, 2, 3, 4, 5, 6, or 7) amino acid residues are A mutation comprising a mutation selected from Ala14Pro, Ser19Arg, Ile20Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu, wherein the glutamic acid at position 1 has been changed to aspartic acid, comprising or essentially consisting of SEQ ID NO: 5. The use of an amino acid sequence comprising:

In another preferred embodiment, the present invention provides a method for preparing a bivalent polypeptide, wherein the amino acid residue is
Glu1Asp,
Glu1Asp, Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Glu1Asp, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Glu1Asp, Gly78Leu, Ala83Arg, Asp85Glu, and Arg105Gln;
Glu1Asp, Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Glu1Asp, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Glu1Asp, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54Asp,
Glu1Asp and Gly54Asp,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Ala83Arg;
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Asp85Glu,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Arg105Gln,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Asp85Glu,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Arg105Gln,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Asp85Glu, and Arg105Gln, or Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Ag, ArA85Ag, ArA85Ag, ArA85Ag
The use of an amino acid sequence comprising or consisting essentially of SEQ ID NO: 5 mutated.

  The invention also relates to the use of three inventive amino acid sequences and / or Nanobodies® (as described above) in preparing trivalent polypeptides. A method for preparing a trivalent polypeptide comprises linking the amino acid sequence of the invention and / or Nanobody®, optionally via one or two linkers.

In a preferred embodiment, the present invention provides a method for preparing a trivalent polypeptide comprising the steps of: a) SEQ ID NO: 60 to SEQ ID NO: 76;
b) an amino acid sequence having only one amino acid difference from one of SEQ ID NO: 60 to SEQ ID NO: 76, preferably only two, more preferably only one;
i) the amino acid sequence has glutamine (Gln, Q) at position 105 (the position is determined by Kabat numbering);
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) (As defined herein) relates to the use of three (preferably identical) amino acid sequences selected from amino acid sequences. The method of preparing a trivalent polypeptide comprises linking the above amino acid sequences, optionally via one or two linkers.

  In a preferred embodiment, the three amino acid sequences used in preparing the trivalent polypeptide comprise or consist essentially of one of SEQ ID NO: 60-SEQ ID NO: 76.

In another preferred aspect, the invention provides a method of preparing a trivalent polypeptide comprising the steps of: a) SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76;
b) only three, preferably only two and more preferably only one amino acid difference from one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75 and SEQ ID NO: 76 An amino acid sequence having
i) the amino acid sequence has glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and / or arginine (Arg, R) at position 83 (the above positions are determined by Kabat numbering; Desired),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) (As defined herein) relates to the use of three (preferably identical) amino acid sequences selected from amino acid sequences. The method of preparing a trivalent polypeptide comprises linking the above amino acid sequences, optionally via one or two linkers.

In another preferred aspect, the invention provides a method of preparing a trivalent polypeptide comprising the steps of: a) SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76;
b) only three, preferably only two and more preferably only one amino acid difference from one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75 and SEQ ID NO: 76 An amino acid sequence having
i) the amino acid sequence has glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and arginine (Arg, R) at position 83 (the above positions are determined by Kabat numbering) ),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) (As defined herein) relates to the use of three (preferably identical) amino acid sequences selected from amino acid sequences. The method of preparing a trivalent polypeptide comprises linking the above amino acid sequences, optionally via one or two linkers.

  In a preferred embodiment, the three amino acid sequences used in preparing the trivalent polypeptide are one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76. Or consists essentially of.

In another preferred embodiment, the present invention provides a method for preparing a trivalent polypeptide comprising the steps of: a) SEQ ID NO: 65 and SEQ ID NO: 76;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NO: 65 and SEQ ID NO: 76,
i) the amino acid sequence is aspartic acid (Asp, D) at position 54, glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and / or arginine (Arg, R) at position 83; (The position is determined by Kabat numbering),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) (As defined herein) relates to the use of three (preferably identical) amino acid sequences selected from amino acid sequences. The method of preparing a trivalent polypeptide comprises linking the above amino acid sequences, optionally via one or two linkers.

In another preferred embodiment, the present invention provides a method for preparing a trivalent polypeptide comprising the steps of: a) SEQ ID NO: 65 and SEQ ID NO: 76;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NO: 65 and SEQ ID NO: 76,
i) the amino acid sequence has aspartic acid (Asp, D) at position 54, glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and arginine (Arg, R) at position 83; (The above position is obtained by Kabat numbering)
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) (As defined herein) relates to the use of three (preferably identical) amino acid sequences selected from amino acid sequences. The method of preparing a trivalent polypeptide comprises linking the above amino acid sequences, optionally via one or two linkers.

  In a preferred embodiment, the three amino acid sequence used in preparing the trivalent polypeptide comprises or consists essentially of SEQ ID NO: 65. In another preferred embodiment, the three amino acid sequence used in preparing the trivalent polypeptide comprises or consists essentially of SEQ ID NO: 76.

In another preferred embodiment, the present invention provides a method for preparing a trivalent polypeptide comprising the steps of: a) SEQ ID NO: 146 to SEQ ID NO: 153;
b) an amino acid sequence having only one, preferably two, more preferably only one amino acid difference from one of SEQ ID NOs: 146 to 153,
i) the amino acid sequence comprises proline (Pro, P) at position 14, arginine (Arg, R) at position 19, leucine (Leu, L) at position 20 and / or leucine (Leu, L) at position 108 Have (the above positions are determined by Kabat numbering),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) (As defined herein) relates to the use of three (preferably identical) amino acid sequences selected from amino acid sequences. The method of preparing a trivalent polypeptide comprises linking the above amino acid sequence to at least one further amino acid sequence, optionally via one or two linkers.

  In a preferred embodiment, the amino acid sequence used in preparing the trivalent polypeptide comprises or consists essentially of one of SEQ ID NOs: 146-153.

In another preferred aspect, the present invention provides a method for preparing a trivalent polypeptide comprising the steps of: a) SEQ ID NOs: 146-149 and SEQ ID NOs: 151-153;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153,
i) the amino acid sequence comprises proline (Pro, P) at position 14, arginine (Arg, R) at position 19, leucine (Leu, L) at position 20, and / or leucine (Leu, L) at position 108 Further having arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85, and / or glutamine (Gln, Q) at position 105 (the position is determined by Kabat numbering),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) (As defined herein) relates to the use of three (preferably identical) amino acid sequences selected from amino acid sequences. The method of preparing a trivalent polypeptide comprises linking the above amino acid sequence to at least one further amino acid sequence, optionally via one or two linkers.

In another preferred aspect, the present invention provides a method for preparing a trivalent polypeptide comprising the steps of: a) SEQ ID NOs: 146-149 and SEQ ID NOs: 151-153;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153,
i) the amino acid sequence is
SEQ ID NO: 146 (this amino acid sequence preferably has glutamine (Gln, Q) at position 105);
SEQ ID NO: 147 (this amino acid sequence preferably has arginine (Arg, R) at position 83 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 148 (this amino acid sequence preferably has glutamic acid (Glu, E) at position 85 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 149 (this amino acid sequence preferably has arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 151 (this amino acid sequence preferably has arginine (Arg, R) at position 83);
SEQ ID NO: 152 (this amino acid sequence preferably has glutamic acid (Glu, E) at position 85);
SEQ ID NO: 153 (this amino acid sequence preferably has arginine (Arg, R) at position 83 and glutamic acid (Glu, E) at position 85)
The amino acid sequence has a proline (Pro, P) at position 14 and an arginine (Arg, R) at position 19, as in the case of having only 3, preferably 2, and more preferably only 1 amino acid difference. Leucine (Leu, L) at position 20 and / or leucine (Leu, L) at position 108, arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85, and / or position 105 Has glutamine (Gln, Q) (the position is determined by Kabat numbering),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) (As defined herein) relates to the use of three (preferably identical) amino acid sequences selected from amino acid sequences. The method of preparing a trivalent polypeptide comprises linking the above amino acid sequence to at least one further amino acid sequence, optionally via one or two linkers.

  In a preferred embodiment, the amino acid sequence used in preparing the trivalent polypeptide comprises or consists essentially of one of SEQ ID NOs: 146-149 and SEQ ID NOs: 151-153.

  In another preferred aspect, the invention provides a method of preparing one or more (eg, 2, 3, 4, 5, 6, 7, 8, or 9, 10, 11, or 12) amino acid residues are mutated from Val5Leu, Ala14Pro, Ser19Arg, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and A1 selected from Gly108Leu. It relates to the use of three (preferably identical) amino acid sequences comprising or consisting essentially of SEQ ID NO: 5.

  In another preferred aspect, the invention provides one or more (eg, 2, 3, 4, 5, 6, 7, 8, or 9) in preparing a trivalent polypeptide. Comprises or consists essentially of SEQ ID NO: 5 having a mutation selected from Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp. (Preferably identical) amino acid sequences.

  In another preferred embodiment, the present invention provides that the method of preparing a trivalent polypeptide comprises the step of preparing one or more (eg, 2, 3 or 4) amino acid residues from Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu. The present invention relates to the use of three (preferably identical) amino acid sequences comprising or consisting essentially of SEQ ID NO: 5, having undergone a selected mutation.

  In another preferred embodiment, the present invention provides a method for preparing a trivalent polypeptide, wherein one or more (eg, 2, 3, 4, 5, 6, or 7) amino acid residues are For the use of three (preferably identical) amino acid sequences comprising or consisting essentially of SEQ ID NO: 5, mutated selected from Ala14Pro, Ser19Arg, Ile20Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu. .

In another preferred embodiment, the present invention provides a method for preparing a trivalent polypeptide, comprising the steps of:
Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Gly78Leu, Ala83Arg, Asp85Glu, and Arg105Gln,
Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54Asp,
Gly54Asp,
Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Ala83Arg,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Asp85Glu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Arg105Gln,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Asp85Glu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Arg105Gln,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Asp85Glu, and Arg105Gln,
It relates to the use of three (preferably identical) amino acid sequences comprising or consisting essentially of SEQ ID NO: 5, mutated from Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, Asp85Glu, and Arg105Gln.

  In another preferred aspect, the invention relates to the use of SEQ ID NO: 62 in preparing SEQ ID NO: 77. A method for preparing a multivalent polypeptide comprises linking the amino acid sequence having SEQ ID NO: 62 with an amino acid sequence having at least two additional SEQ ID NO: 62 via a 15GS (SEQ ID NO: 128) linker.

  In another preferred aspect, the invention relates to the use of SEQ ID NO: 65 in preparing SEQ ID NO: 78. A method for preparing a multivalent polypeptide comprises linking the amino acid sequence having SEQ ID NO: 65 with at least two additional amino acid sequences having SEQ ID NO: 65 via a 15GS (SEQ ID NO: 128) linker.

  In another preferred aspect, the invention relates to the use of SEQ ID NO: 76 in preparing SEQ ID NO: 79. A method for preparing a multivalent polypeptide comprises linking the amino acid sequence having SEQ ID NO: 76 with at least two additional amino acid sequences having SEQ ID NO: 76 via a 15GS (SEQ ID NO: 128) linker.

  In another preferred aspect, the invention relates to the use of SEQ ID NO: 75 in preparing SEQ ID NO: 158. A method for preparing a multivalent polypeptide comprises linking the amino acid sequence having SEQ ID NO: 75 with at least two additional amino acid sequences having SEQ ID NO: 75 via a 15GS (SEQ ID NO: 128) linker.

  In another preferred aspect, the invention relates to the use of SEQ ID NO: 147 in preparing SEQ ID NO: 159. A method for preparing a multivalent polypeptide involves linking the amino acid sequence having SEQ ID NO: 147 with at least two additional amino acid sequences having SEQ ID NO: 147 via a 15GS (SEQ ID NO: 128) linker.

  In another preferred aspect, the invention relates to the use of SEQ ID NO: 149 in preparing SEQ ID NO: 160. A method of preparing a multivalent polypeptide comprises linking the amino acid sequence having SEQ ID NO: 149 with at least two additional amino acid sequences having SEQ ID NO: 149 via a 15GS (SEQ ID NO: 128) linker.

  In another preferred aspect, the invention relates to the use of SEQ ID NO: 153 in preparing SEQ ID NO: 161. A method for preparing a multivalent polypeptide comprises linking the amino acid sequence having SEQ ID NO: 153 with at least two additional amino acid sequences having SEQ ID NO: 153 via a 15GS (SEQ ID NO: 128) linker.

  In another preferred embodiment, the present invention relates to the use of an amino acid sequence selected from the above wherein the amino acid at position 1 (glutamic acid) is changed to aspartic acid in preparing a trivalent polypeptide.

In this regard, the present invention provides a method for preparing a trivalent polypeptide comprising the steps of: a) SEQ ID NO: 138 to SEQ ID NO: 141 and SEQ ID NO: 154 to SEQ ID NO: 157;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 138 to 141 and 154 to 157,
i) the amino acid sequence has aspartic acid (Asp, D) at position 1 (the position is determined by Kabat numbering);
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or whose amino acid sequence is the same, about the same, or higher in potency (as compared to an amino acid sequence having no three, two or one amino acid differences) (As defined herein) relates to the use of an amino acid sequence selected from amino acid sequences. A method for preparing a trivalent polypeptide comprises linking the above amino acid sequence to at least two additional amino acid sequences, optionally via one or two linkers.

  In a preferred embodiment, the amino acid sequence used in preparing the trivalent polypeptide comprises or consists essentially of SEQ ID NO: 138. In another preferred embodiment, the amino acid sequence used in preparing the trivalent polypeptide comprises or consists essentially of SEQ ID NO: 139. In another preferred embodiment, the amino acid sequence used in preparing the trivalent polypeptide comprises or consists essentially of SEQ ID NO: 140. In another preferred embodiment, the amino acid sequence used in preparing the trivalent polypeptide comprises or consists essentially of SEQ ID NO: 141. In another preferred embodiment, the amino acid sequence used in preparing the trivalent polypeptide comprises or consists essentially of one of SEQ ID NOs: 154-157.

  In another preferred aspect, the invention provides the use of an amino acid sequence comprising or consisting essentially of SEQ ID NO: 5, wherein glutamic acid at position 1 has been changed to aspartic acid in preparing a trivalent polypeptide. About.

  In another preferred aspect, the invention provides the use of an amino acid sequence comprising or consisting essentially of SEQ ID NO: 62 wherein glutamic acid at position 1 has been changed to aspartic acid in preparing a trivalent polypeptide. About.

  In another preferred aspect, the invention provides the use of an amino acid sequence comprising or consisting essentially of SEQ ID NO: 65, wherein glutamic acid at position 1 has been changed to aspartic acid in preparing a trivalent polypeptide. About.

  In another preferred aspect, the invention provides the use of an amino acid sequence comprising or consisting essentially of SEQ ID NO: 76, wherein glutamic acid at position 1 has been changed to aspartic acid in preparing a trivalent polypeptide. About.

  In another preferred aspect, the invention provides the use of an amino acid sequence comprising or consisting essentially of SEQ ID NO: 75 wherein glutamic acid at position 1 has been changed to aspartic acid in preparing a trivalent polypeptide. About.

  In another preferred aspect, the invention provides the use of an amino acid sequence comprising or consisting essentially of SEQ ID NO: 147, wherein glutamic acid at position 1 has been changed to aspartic acid in preparing a trivalent polypeptide. About.

  In another preferred aspect, the invention provides the use of an amino acid sequence comprising or consisting essentially of SEQ ID NO: 149, wherein glutamic acid at position 1 has been changed to aspartic acid in preparing a trivalent polypeptide. About.

  In another preferred aspect, the invention provides for the use of an amino acid sequence comprising or consisting essentially of SEQ ID NO: 153, wherein glutamic acid at position 1 has been changed to aspartic acid in preparing a trivalent polypeptide. About.

  In another preferred aspect, the invention provides a method of preparing one or more (eg, 2, 3, 4, 5, 6, 7, 8, or 9, (10, 11, or 12) amino acid residues are mutated from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54A selected from Gly54A. 1, wherein the glutamic acid at position 1 has been changed to aspartic acid, comprising or consisting essentially of SEQ ID NO: 5.

  In another preferred aspect, the invention provides one or more (eg, 2, 3, 4, 5, 6, 7, 8, or 9) in preparing a trivalent polypeptide. Has undergone a mutation selected from Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp, and the glutamic acid at position 1 has been changed to aspartic acid. 5. Use of an amino acid sequence comprising or consisting essentially of 5.

  In another preferred embodiment, the present invention provides that the method of preparing a trivalent polypeptide comprises the step of preparing one or more (eg, 2, 3 or 4) amino acid residues from Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu. The present invention relates to the use of an amino acid sequence comprising or consisting essentially of SEQ ID NO: 5, which has undergone a selected mutation and wherein the glutamic acid at position 1 has been changed to aspartic acid.

  In another preferred embodiment, the present invention provides a method for preparing a trivalent polypeptide, wherein one or more (eg, 2, 3, 4, 5, 6, or 7) amino acid residues are A mutation comprising a mutation selected from Ala14Pro, Ser19Arg, Ile20Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu, wherein the glutamic acid at position 1 has been changed to aspartic acid, including or consisting essentially of SEQ ID NO: 5. The use of an amino acid sequence comprising:

In another preferred embodiment, the present invention provides a method for preparing a trivalent polypeptide, comprising the steps of:
Glu1Asp,
Glu1Asp, Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Glu1Asp, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Glu1Asp, Gly78Leu, Ala83Arg, Asp85Glu, and Arg105Gln;
Glu1Asp, Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Glu1Asp, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Glu1Asp, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54Asp,
Glu1Asp and Gly54Asp,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Ala83Arg;
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Asp85Glu,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Arg105Gln,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Asp85Glu,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Arg105Gln,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Asp85Glu, and Arg105Gln, or Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Ag, ArA85Ag, ArA85Ag, ArA85Ag
The use of an amino acid sequence comprising or consisting essentially of SEQ ID NO: 5 mutated.

  In another preferred aspect, the invention relates to the use of SEQ ID NO: 138 in preparing SEQ ID NO: 142. A method for preparing a multivalent polypeptide comprises linking the amino acid sequence having SEQ ID NO: 138 with at least two additional amino acid sequences (preferably SEQ ID NO: 5) via a 15GS (SEQ ID NO: 128) linker.

  In another preferred aspect, the invention relates to the use of SEQ ID NO: 139 in preparing SEQ ID NO: 143. A method for preparing a multivalent polypeptide comprises linking the amino acid sequence having SEQ ID NO: 139 with at least two additional amino acid sequences (preferably SEQ ID NO: 62) via a 15GS (SEQ ID NO: 128) linker.

  In another preferred aspect, the invention relates to the use of SEQ ID NO: 140 in preparing SEQ ID NO: 144. A method for preparing a multivalent polypeptide involves linking the amino acid sequence having SEQ ID NO: 140 with at least two additional amino acid sequences (preferably SEQ ID NO: 65) via a 15GS (SEQ ID NO: 128) linker.

  In another preferred aspect, the invention relates to the use of SEQ ID NO: 154 in preparing SEQ ID NO: 162. A method for preparing a multivalent polypeptide comprises linking the amino acid sequence having SEQ ID NO: 154 with at least two additional amino acid sequences (preferably SEQ ID NO: 75) via a 15GS (SEQ ID NO: 128) linker.

  In another preferred aspect, the invention relates to the use of SEQ ID NO: 155 in preparing SEQ ID NO: 163. A method for preparing a multivalent polypeptide involves linking the amino acid sequence having SEQ ID NO: 155 with at least two additional amino acid sequences (preferably SEQ ID NO: 147) via a 15GS (SEQ ID NO: 128) linker.

  In another preferred aspect, the invention relates to the use of SEQ ID NO: 156 in preparing SEQ ID NO: 164. A method for preparing a multivalent polypeptide comprises linking the amino acid sequence having SEQ ID NO: 156 with at least two additional amino acid sequences (preferably SEQ ID NO: 149) via a 15GS (SEQ ID NO: 128) linker.

  In another preferred aspect, the invention relates to the use of SEQ ID NO: 157 in preparing SEQ ID NO: 165. A method for preparing a multivalent polypeptide involves linking an amino acid sequence having SEQ ID NO: 157 with at least two additional amino acid sequences (preferably SEQ ID NO: 153) via a 15GS (SEQ ID NO: 128) linker.

  The at least one amino acid sequence or Nanobody® is directed to a first antigen (ie, to protein F of hRSV) and the at least one amino acid sequence or Nanobody® is directed to a second ( That is, a polypeptide of the invention containing at least two amino acid sequences and / or Nanobodies® that is directional to an antigen (different from hRSV protein F) is a “multispecific” of the invention. Also referred to as polypeptides, the amino acid sequence or Nanobody® present in such polypeptides is also referred to herein as being in a “multispecific format”. Thus, for example, a “bispecific” polypeptide of the invention comprises at least one amino acid sequence or Nanobody® of the invention directed to a first antigen (ie, protein F of hRSV), (Ie, different from protein F of hRSV) is a polypeptide comprising at least one additional amino acid sequence or Nanobody® directed to an antigen, wherein the “trispecific” polypeptide of the invention comprises: At least one amino acid sequence or Nanobody® of the invention directed to a first antigen (ie, hRSV protein F) and a second (ie, different from hRSV protein F) antigen At least one additional amino acid sequence or Nanobody® having the third (ie, hRSV Park protein differs from both the F and the second antigen) is a polypeptide comprising at least one additional amino acid sequences or Nanobodies directed against the antigen.

  Thus, in its simplest form, a bispecific polypeptide of the invention comprises a first amino acid sequence or Nanobody® of the invention directed to protein F of hRSV and a second antigen A directional second amino acid sequence or Nanobody®, wherein the first and second amino acid sequences or Nanobody® are optionally linkers (as defined herein). A bivalent polypeptide of the invention (as defined herein) which can be linked via a sequence, the simplest form of the trispecific polypeptide of the invention being directed to protein F of hRSV. A first amino acid sequence or Nanobody (registered trademark) of the present invention, a second amino acid sequence or Nanobody (registered trademark) having directivity to a second antigen, and a third antigen A third amino acid sequence or Nanobody (registered trademark) having directivity, wherein the first, second and third amino acid sequences or Nanobody (registered trademark) are optionally one or more, particularly two A trivalent polypeptide of the invention (as defined herein) which can be linked via a linker sequence.

  In certain embodiments, a polypeptide of the invention is a trivalent bispecific polypeptide. In its simplest form, the trivalent bispecific polypeptide of the present invention comprises two identical amino acid sequences or Nanobodies® for protein F of hRSV and a third antigen directed to another antigen. Amino acid sequences or Nanobodies®, wherein the first, second and third amino acid sequences or Nanobodies® are optionally linked via one or more, in particular two, linker sequences. The resulting trivalent polypeptide of the invention (as defined herein) may be.

  Preferred, but non-limiting examples of multispecific polypeptides of the invention include at least one amino acid sequence or Nanobody® of the invention and at least one Nanobody® that provides an increased half-life. Including. Some preferred but non-limiting examples of such Nanobodies® include serum proteins such as human serum albumin, thyroxine binding protein, (human) transferrin, fibrinogen, immunoglobulins such as IgG, IgE or IgM. Or Nanobodies® that are directional to one of the other serum proteins listed in WO 04/003019.

  For example, for experiments in mice, Nanobodies® for mouse serum albumin (MSA) can be used, and for pharmaceutical use, Nanobodies® for human serum albumin can be used.

  In another embodiment of the invention, the at least one (human) serum protein is (human) serum albumin, (human) serum immunoglobulin, (human) thyroxine binding protein, (human) transferrin, (human) fibrinogen and the like. Or a polypeptide construct as described above.

  Thus, in certain embodiments, the polypeptide of the invention comprises a first amino acid sequence or Nanobody® of the invention for protein F of hRSV and a second amino acid sequence orientated to (human) serum albumin. And a bispecific polypeptide, wherein the first and second amino acid sequences or Nanobodies® described above can be optionally linked via a linker sequence.

  In another particular embodiment, the polypeptide of the invention comprises two identical amino acid sequences or Nanobodies of the invention for protein F of hRSV and a third amino acid directed to (human) serum albumin Or the first, second and third amino acid sequences or Nanobodies® described above may optionally be linked via one or more, in particular two, linker sequences. It is a trivalent bispecific polypeptide.

  In another particular embodiment, the polypeptide of the invention comprises three identical amino acid sequences or Nanobodies of the invention for protein F of hRSV and a fourth amino acid directed to (human) serum albumin. Or the first, second, third and fourth amino acid sequences or Nanobody® described above, optionally one or more, in particular two or three linker sequences. Is a tetravalent bispecific polypeptide that can be linked through

  According to a specific, but non-limiting embodiment of the invention, the polypeptide of the invention is directed to at least one human serum albumin in addition to one or more amino acid sequences or Nanobodies of the invention. Contains Nanobody®. These Nanobodies® for human serum albumin may be those described comprehensively in the above-cited application by Ablynx N.V. (see, eg, WO 04/062551). Some particularly preferred Nanobodies® that provide an increased half-life and can be used in the polypeptides of the present invention include WO 06/122787 (see Table II and Table III). The disclosed Nanobodies (registered trademark) ALB-1 to ALB-10 are mentioned, and ALB-8 (SEQ ID NO: 62 in WO 06/122787) is particularly preferred.

  In another aspect, the invention comprises or consists essentially of one or more of the amino acid sequences, Nanobodies® and / or polypeptides (or suitable fragments thereof) of the invention, optionally Or a compound or construct, especially a protein or polypeptide (also referred to herein as a "compound of the invention"), further comprising one or more other groups, residues, moieties or binding units. As will be apparent to those skilled in the art from the further disclosure herein, such additional groups, residues, moieties, binding units or amino acid sequences may be incorporated into an amino acid sequence, Nanobody® or polypeptide of the invention. (And / or the compound or construct in which it is present) may or may not have additional functionality, and may alter the properties of the amino acid sequence, Nanobody® and / or polypeptide of the invention. Or it may not be changed.

  Such groups, residues, moieties or linking units can be, for example, chemical groups, residues, moieties (whether themselves biologically and / or pharmacologically active or not). Good). For example, but not limited to, such a group can be used to provide a "derivative" of an amino acid sequence, Nanobody (R) and / or polypeptide of the invention, as described further herein. It may be linked to one or more amino acid sequences, Nanobodies® and / or polypeptides of the invention.

  One or more other groups, including or consisting essentially of one or more derivatives as described herein, optionally linked via one or more linkers, Compounds or constructs that optionally further comprise groups, moieties or linking units are also within the scope of the invention. Preferably, the one or more other groups, residues, moieties or binding units are amino acid sequences.

  In a compound, construct or polypeptide as described above, one or more of the amino acid sequences, Nanobodies® and / or polypeptides of the invention, and one or more groups, residues, moieties or binding units may be , Directly to one another and / or via one or more suitable linkers or spacers. For example, where one or more groups, residues, moieties or linking units are amino acid sequences, the linker is such that the resulting compound, construct or polypeptide is a fusion (protein) or fusion (polypeptide). It may be an amino acid sequence.

  The compound or construct of the present invention is fused at its amino terminus, at its carboxy terminus, or at both its amino and carboxy terminus, to at least one further amino acid sequence, ie the amino acid sequence of the invention as described above, a Nanobody Amino acid sequences, Nanobodies® or polypeptides of the invention may be included to provide a fusion protein comprising a ® or polypeptide and one or more additional amino acid sequences.

  The one or more additional amino acid sequences can be any suitable and / or desired amino acid sequence. The further amino acid sequence may alter or modify, or otherwise affect, the (biological) properties of the amino acid sequence, Nanobody® or polypeptide of the invention. It may be absent, and may or may not have additional functionality added to the amino acid sequences, Nanobodies® or polypeptides of the invention. Preferably, the additional amino acid sequence confers one or more desired properties or functionalities to the amino acid sequence, Nanobody® or polypeptide of the invention.

  Examples of such amino acid sequences will be apparent to those skilled in the art and may generally include all amino acid sequences used in peptide fusions based on conventional antibodies and fragments thereof (including ScFv and single domain antibodies, But not limited to these). See, for example, the review by Holliger and Hudson, Nature Biotechnology, 23, 9, 1126-1136 (2005).

  For example, such amino acid sequences may increase half-life, solubility or absorption, reduce immunogenicity or toxicity, desirably, compared to the amino acid sequences, Nanobodies® or polypeptides of the invention. It may be an amino acid sequence that eliminates or attenuates unwanted side effects and / or imparts other beneficial properties to the compounds of the invention and / or reduces undesired properties of the compounds of the invention. Some non-limiting examples of such amino acid sequences are serum proteins, such as human serum albumin (see, eg, WO 00/27435) or hapten molecules (eg, haptens (eg, hapten recognized as circulating antibodies). See, for example, WO 98/22141)).

  The additional amino acid sequence may also include any desired proteins, polypeptides, antigens, antigenic determinants or epitopes (similar proteins, polypeptides, antigens, antigens to which the amino acid sequences or Nanobodies® of the invention are directed). Determinants or epitopes, or any of different proteins, polypeptides, antigens, antigenic determinants or epitopes, including, but not limited to. For example, the additional amino acid sequence may provide a second binding site directed to a serum protein (eg, another serum protein such as human serum albumin or IgG) so as to provide an increased half-life in serum. it can. Such amino acid sequences include, for example, Nanobodies®, and small peptides and binding proteins described in WO 91/01743, WO 01/45746 and WO 02/076489, and And dAbs described in WO 03/002609 and WO 04/003019. Harmsen et al., Vaccine, 23 (41); 4926-42, 2005, as well as EP 0 368 684 and Ablynx N.V. See also WO 08/043822 and WO 08/068280.

  Such an amino acid sequence may be particularly directed to serum albumin (more specifically, human serum albumin) and / or to IgG (more specifically, human IgG). For example, such an amino acid sequence is an amino acid sequence directed to (human) serum albumin and an amino acid sequence capable of binding to an amino acid residue on (human) serum albumin that is not involved in binding of serum albumin to FcRn. (See, eg, WO 06/0122787) and / or amino acid sequences capable of binding to amino acid residues on serum albumin that do not form the domain III portion of serum albumin (eg, WO 06/0122787) Amino acid sequences having or capable of increasing half-life (see, eg, WO 08/028977); serum albumin from at least one mammal; Especially at least one primate (for example, but not limited to And cross-reactivity with macaque monkeys (eg, especially cynomolgus monkeys (Macaca fascicularis) and / or rhesus monkeys (Macacamulatta)) and baboons (Papio ursinus). Amino acid sequence for human serum albumin (also see WO 08/028977); amino acid sequence capable of binding serum albumin in a pH-independent manner (see eg WO 08/043821) And / or amino acid sequences that are conditional binders (see, for example, WO 08/043822).

According to another embodiment, the one or more additional amino acid sequences comprises one or more parts, fragments or domains of a conventional four-chain antibody (particularly a human antibody) and / or a heavy-chain antibody. obtain. For example, although usually less preferred, the amino acid sequences, Nanobodies® or polypeptides of the present invention can be added to a conventional (preferably human) _VH or _VL domain, optionally also via a linker sequence. Or linked to a natural or synthetic analog of a _VH or _VL domain, including but not limited to other (single) domain antibodies such as the dAbs described by Ward et al. Can be done.

  Thus, in the compounds or constructs of the present invention, the one or more other groups, residues, moieties or binding units described above may comprise a domain antibody, an amino acid sequence suitable for use as a domain antibody, a single domain antibody, Amino acid sequences suitable for use as domain antibodies, "dAbs", amino acid sequences suitable for use as dAbs, or Nanobodies®.

  In one particular aspect of the invention, a compound, construct or polypeptide of the invention comprising at least one amino acid sequence, Nanobody (R) or polypeptide of the invention comprises a corresponding amino acid sequence, Nanobody (R) of the invention. (Trademark) or polypeptides. Some preferred but non-limiting examples of such compounds, constructs and polypeptides will be apparent to those of skill in the art based on the further disclosure herein, e.g., to increase half-life (e.g., PEG An amino acid sequence, Nanobody®, or polypeptide of the invention that has been chemically modified (by at least one); or at least one portion (particularly at least one) that increases the half-life of the amino acid sequence, Nanobody®, or polypeptide of the invention. A single amino acid sequence), a compound of the invention comprising at least one Nanobody®, polypeptide or polypeptide of the invention linked to it. Examples of compounds of the present invention that include such a half-life extending moiety will be apparent to those skilled in the art based on the further disclosure herein, including, but not limited to, one or more compounds of the present invention. Amino acid sequence, Nanobody® or polypeptide of one or more serum proteins or fragments thereof (eg, serum albumin or suitable fragments thereof), or one or more linkages capable of binding serum proteins A compound suitably linked to a unit (eg, a Nanobody® or (single) domain antibody capable of binding serum proteins such as serum albumin, serum immunoglobulins such as IgG or transferrin); The amino acid sequence, Nanobody® or polypeptide is an Fc portion (eg, human Fc) or A compound linked to a suitable moiety or fragment; or one or more small proteins or small peptides to which one or more of the amino acid sequences, Nanobodies® or polypeptides of the invention can bind to serum proteins (Eg, proteins and peptides described in WO 91/01743, WO 01/45746, and WO 02/076489).

Also, at least one amino acid sequence, Nanobody® or polypeptide, optionally via a linker sequence, may contain one or more (preferably human) C _H 1 domains, C _H 2 domains and / or C _H It may be linked to three domains. For example, an amino acid sequence, Nanobody®, or polypeptide that links to a suitable C _H 1 domain is similar to, for example, a conventional Fab fragment or F (ab ′) ₂ fragment, but one of the conventional V _H domains, Or (in the case of the F (ab ') ₂ fragment) to produce an antibody fragment / structure in which one or both of the amino acid sequences, Nanobodies® or polypeptides of the invention have been replaced (suitable light chains Can be used with Further, two amino acid sequences or Nanobodies can result (optionally via a linker) construct half-life in vivo in conjunction with the C H ₃ domain is increased.

According to one particular aspect, one or more of the amino acid sequences, Nanobodies® or polypeptides of the invention comprises one or more constant domains (eg used as part of an Fc portion / one of Fc Conferring an amino acid sequence, Nanobody (R) or polypeptide of the invention to the amino acid sequence, Nanobody (R) or polypeptide of the invention; And / or linked (optionally via a suitable linker or hinge region) to one or more antibody moieties, fragments or domains that may confer the ability to bind to one or more Fc receptors. For example, and without limitation, for this purpose, the one or more additional amino acid sequences may be a heavy chain antibody (as described herein) and more preferably a conventional human four-chain antibody. may contain one or more of the _{C H} 2 domain and / or _{C H} 3 domain of an antibody such as derived from, and / or e.g. IgG (e.g. IgG1, IgG2, IgG3 or IgG4), IgE, or IgA , IgD or IgM (part of) an Fc region derived from another human Ig. For example WO 94/04678, the camelid _{V HH} domain or a humanized derivative thereof (i.e., a Nanobody (TM)) describes a heavy chain antibody comprising, here, camelid _C H 2 The domain and / or the _CH3 domain has been replaced by a human _CH2 domain and a _CH3 domain. This results in an immunoglobulin consisting of two heavy chains, each containing Nanobody® and a human C _H 2 and C _H 3 domain (without the C _H 1 domain), respectively. has the effector function conferred by the C H ₂ domain and C _{H 3} domains, can function in the absence of any light chain. Other amino acid sequences that can be suitably linked to the amino acid sequences, Nanobodies® or polypeptides of the invention to provide effector functions will be apparent to those of skill in the art, and can be selected based on the desired effector function (s). Can be. For example, WO 04/058820, WO 99/42077, WO 02/056910, and WO 05/017148, and an overview of Holliger and Hudson (ibid.), And WO 09/42078. 068628. Conjugation of an amino acid sequence, Nanobody (R) or polypeptide of the invention to an Fc moiety may also result in an increase in half-life compared to the corresponding amino acid sequence, Nanobody (R) or polypeptide of the invention. . For some applications, the use of Fc portions and / or constant domains (ie, C _H 2 and / or C _H 3 domains) that provide an increased half-life without any biologically important effector functions is also suitable. Or may be more preferred. Other suitable constructs comprising one or more amino acid sequences, Nanobodies® or polypeptides and one or more constant domains with increased half-life in vivo will be apparent to those skilled in the art, for example, amino acid sequence linked to the C _{H 3} domain via a linker sequence, may include Nanobodies or polypeptides. In general, any fusion protein or derivative with an increased half-life preferably has a molecular weight of more than 50 kD, ie a cut-off value for renal absorption.

  In another specific, but non-limiting embodiment, one or more of the amino acid sequences, Nanobodies®, or polypeptides of the invention is used to form a compound of the invention (ie, Natural, synthetic or semi-synthetic constant domains (or similar) that have a reduced (or essentially no) tendency to self-associate with a dimer (as compared to the naturally occurring constant domains of a four-chain antibody) (Variants, variants, mutants, parts or fragments) (optionally via a suitable linker or hinge region). Such monomeric (ie, non-self associated) Fc chain variants or fragments thereof will be apparent to those of skill in the art. For example, Helm et al., J Biol Chem 1996 271 7494, describe monomeric Fc chain variants that can be used in the polypeptide chains of the invention.

  Also, such monomeric Fc chain variants are still capable of binding complement or the associated Fc receptor (s) (depending on the Fc portion from which they are derived), and Preferably they are such that they still have some or all (or still at a reduced level suitable for the intended use) of the effector function of the Fc moiety from which they are derived. Alternatively, in such polypeptide chains of the invention, the monomer Fc chain can be used to increase the half-life of the polypeptide chain, where the monomer Fc chain has an effector function. It may not have, or may not have essentially.

  In general, an amino acid sequence, Nanobody®, or polypeptide of the invention (or a compound, construct, or composition comprising the same) that has an increased half-life is a corresponding amino acid sequence, Nanobody®, or polypeptide of the invention. It preferably has a half-life that is at least 1.5 times, preferably at least 2 times (eg at least 5 times), for example at least 10 times, or more than 20 times greater than its own half life. For example, an amino acid sequence, Nanobody (R), compound, construct or polypeptide of the invention having an increased half-life can have a 1-amino acid sequence, Nanobody (R) or polypeptide as compared to the corresponding amino acid sequence, Nanobody (R) or polypeptide itself. It may have an increased serum half-life of more than 2 hours, preferably more than 2 hours, more preferably more than 6 hours (eg more than 12 hours), or even more than 24 hours, 48 hours or 72 hours.

  In a preferred, but non-limiting embodiment of the invention, such an amino acid sequence, Nanobody®, compound, construct or polypeptide of the invention comprises at least about 12 hours, preferably at least 24 hours, more preferably at least 24 hours. A serum half-life in humans of 48 hours, even more preferably at least 72 hours or more. For example, a compound or polypeptide of the present invention can be administered for at least 5 days (eg, about 5-10 days), preferably at least 9 days (eg, about 9-14 days), more preferably at least about 10 days (eg, about 10 days). -15 days), or at least about 11 days (eg, about 11-16 days), more preferably at least about 12 days (eg, about 12-18 days or more), or more than 14 days (eg, about 14-19 days) )).

  Also, additional amino acid sequences may be synthesized during synthesis (eg, to provide a preform, proform, or preproform of a polypeptide of the invention, depending on the host cell used to express the polypeptide of the invention). To form a signal sequence or leader sequence that directs secretion of the amino acid sequence, Nanobody® or polypeptide of the present invention from the host cell.

  Also, additional amino acid sequences may direct the amino acid sequence, Nanobody® or polypeptide of the invention to a particular organ, tissue, cell, or portion or compartment of a cell and / or penetrate them. Or penetrates and / or allows the amino acid sequence, Nanobody® or polypeptide of the invention to penetrate or cross biological barriers, such as cell membranes, cell layers such as epithelial cell layers, etc. Can form a sequence or signal. Suitable examples of such amino acid sequences will be clear to the skilled person, for example and without limitation, the "Pepttrans" vector described above, the sequence described by Cardinale et al., And for example WO 94/02610. WO 95/22618, U.S. Patent No. 7,004,940, WO 03/014960, WO 99/07414; WO 05/01690; EP 1512696; Cattaneo, A. & Biocca, S. (1997) Intracellular Antibodies: Development and Applications. Landes and Springer-Verlag, and Kontermann, Methods 34, (2004), 163-170, which are described in further references therein. Such a so-called “intracellular antibody” can be used to express or produce Nanobodies® and polypeptides of the invention. That itself include known amino acid sequences and antibody fragments.

  Such a protein, polypeptide, compound or construct may be in essentially isolated form (as defined herein).

  A compound or polypeptide of the invention generally comprises one or more amino acid sequences of the invention, Nanobodies (optionally via one or more suitable linkers) to provide a compound or polypeptide of the invention. ®, monovalent constructs, and / or polypeptides can be prepared by a method that includes at least the step of suitably linking one or more additional groups, residues, moieties, or binding units. The polypeptide of the present invention can also be prepared by a method generally comprising at least the steps of preparing a nucleic acid encoding the polypeptide of the present invention, suitably expressing the nucleic acid, and recovering the expressed polypeptide of the present invention. Can be prepared. Such methods can be performed in a manner known per se, and will be apparent to those skilled in the art, for example, based on the methods and techniques further described herein.

  Suitable spacers or linkers for use in multivalent and / or multispecific polypeptides or constructs will be apparent to those of skill in the art, and generally include any linker or linker used in the art to join amino acid sequences. It may be a spacer. Preferably, the linker or spacer is suitable for use in constructing a protein or polypeptide intended for pharmaceutical use.

Some particularly preferred spacers include spacers and linkers used in the art to link antibody fragments or antibody domains. These include, for example, linkers used in the art to construct diabodies or ScFv fragments, as well as the linkers mentioned in the general background art referred to above (but in this regard, diabodies and ScFvs). In fragments, the linker sequence used must be of a length, degree of flexibility, and other properties that allow the associated _VH and _VL domains to combine to form a complete antigen-binding site. Although each amino acid sequence or Nanobody (registered trademark) alone forms a complete antigen-binding site, the length or flexibility of the linker used in the polypeptide of the present invention is particularly important. Note that there are no restrictions).

For example, the linker may be a suitable amino acid sequence, specifically an amino acid sequence having 1 to 50, preferably 1 to 30, for example 1 to 10 amino acid residues. Some preferred examples of such amino acid sequences, for example as described in WO 99/42077 _(gly 4 _{ser) 3,} or _(gly 3 _{ser 2) 3} such as for example _(gly x _ser y) Gly-ser linkers in the _z- form, and hinge-like regions such as the hinge region of native heavy chain antibodies, or similar sequences (such as those described in WO 94/04678).

  Some other particularly preferred linkers are polyalanines (eg, AAA), as well as the linkers mentioned in Table A-7, of which GS15 is particularly preferred.

  In general, other suitable linkers include organic compounds or polymers, especially organic compounds or polymers suitable for use in proteins for pharmaceutical use. For example, poly (ethylene glycol) moieties have been used to link antibody domains. See, for example, WO 04/081026.

  The length, degree of flexibility and / or other properties of the linker (s) used (but are not critical as they are usually the same as for linkers used for ScFv fragments) are It is within the scope of the present invention that any property of the polypeptides of the invention, including but not limited to affinity, specificity or binding activity of hRSV to protein F or one or more other antigens, can be affected. Contained within. One of skill in the art, based on the disclosure herein, may optionally perform some limited routine experimentation to determine the optimal linker (s) for use with a particular polypeptide of the invention. Could be determined.

  The linker (s) used confer one or more other advantageous properties or functionalities to the polypeptide of the invention and / or (e.g., an amino acid sequence of the invention, a Nanobody®, It is also within the scope of the present invention to provide one or more sites for the formation of derivatives and / or attachment of functional groups (as described herein for derivatives of compounds and polypeptides). For example, linkers containing one or more charged amino acid residues can provide improved hydrophilicity, whereas linkers that form or contain small epitopes or tags can be detected, identified and / or identified. Alternatively, it can be used for purification purposes. Again, the skilled artisan will, based on the disclosure herein, optionally perform some limited routine experimentation to determine the optimal linker for use with a particular polypeptide of the invention. Could be determined.

  Finally, if more than one linker is used in the polypeptide of the invention, these linkers may be the same or different. Again, the skilled artisan will, based on the disclosure herein, optionally perform some limited routine experimentation to determine the best linker to use for a particular polypeptide of the invention. Could be determined.

  Usually, to facilitate expression and production, the polypeptides of the present invention are linear polypeptides. However, the invention is not so limited in the broadest sense. For example, if the polypeptide of the invention comprises three or more amino acid sequences or Nanobodies (R), each "arm" has an amino acid sequence or Nanobodies (R) to obtain a "star-shaped" construct. It is possible to link them by using a linker with three or more "arms" linking to the same. Although usually less preferred, it is also possible to use circular constructs.

  As will also be apparent from the disclosure herein, a portion or fragment of an amino acid sequence, Nanobody® or polypeptide of the invention, as defined herein, or a combination of two or more portions or fragments. In particular, the amino acid sequence of SEQ ID NO: 60 to SEQ ID NO: 76, SEQ ID NO: 138 to SEQ ID NO: 141 and SEQ ID NO: 146 to SEQ ID NO: 157, or SEQ ID NO: 77 to SEQ ID NO: 79, SEQ ID NO: 142 to SEQ ID NO: 145, and SEQ ID NO: 158 to Use of a portion or fragment of the polypeptide of SEQ ID NO: 165 is also within the scope of the invention. Thus, according to one embodiment of the invention, the terms “amino acid sequence of the invention”, “Nanobody® of the invention” and “polypeptide of the invention” in the broadest sense refer to such a moiety or Fragments are also included.

  Generally, such portions or fragments of the amino acid sequences, Nanobodies® or polypeptides of the invention (including variants thereof as defined herein) comprise the corresponding full length amino acid sequences or Nanobodies of the invention. One or more amino acid residues at the N-terminus, one or more amino acid residues at the C-terminus, one or more consecutive internal amino acid residues, or any Has an amino acid sequence that has been deleted and / or has been removed.

Parts or fragments, as defined in the above (further described herein herein, (actual or apparent) K _D values (actual or apparent) K _A value k _on rate and And / or so as to be able to bind antigen site II on protein F of hRSV with an affinity (appropriately measured and / or expressed as a k _off rate or alternatively as an IC ₅₀ value) It is preferred that

In particular, amino acid sequences, Nanobodies®, polypeptides, and parts or fragments are preferably
Binds to protein F of hRSV with a dissociation constant (K _D ) of 1000 nM to 1 nM or less, preferably 100 nM to 1 nM or less, more preferably 10 nM to 1 nM or less, and / or 10 ⁴ M ⁻¹ s ⁻¹ to about Protein of hRSV with a k _on rate of 10 ⁷ M ⁻¹ s ⁻¹ , preferably 10 ⁵ M ⁻¹ s ⁻¹ to 10 ⁷ M ⁻¹ s ⁻¹ , more preferably about 10 ⁶ M ⁻¹ s ⁻¹ or more. F and / or 10 ⁻² s ⁻¹ (t _1/2 = 0.69 s) to 10 ⁻⁴ s ⁻¹ (substantially irreversible complex in which t _1/2 is several days. In this case), it is preferable that the protein binds to hRSV protein F at a k _off rate of preferably 10 ⁻³ s ⁻¹ to 10 ⁻⁴ s ⁻¹ or less.

  The affinity of a portion or fragment for protein F of hRSV can be determined using methods known per se, for example, the assays described herein.

  Also, such portions or fragments typically have the efficacy and / or efficacy of neutralizing hRSV as defined herein.

  Any part or fragment is at least 10 contiguous amino acid residues, preferably at least 20 contiguous amino acid residues of the amino acid sequence of the corresponding full-length amino acid sequence, Nanobody® or polypeptide of the present invention. More preferably, it contains at least 30 contiguous amino acid residues, such as at least 40 contiguous amino acid residues.

  Also, any part or fragment is preferably linked to at least one of the CDRs (preferably at least CDR3 or a part thereof), preferably linked by a suitable framework sequence (s) or at least a part thereof. Preferably, it comprises one other CDR (ie CDR1 or CDR2) or at least a part thereof. More preferably, any part or fragment is at least one (preferably at least CDR3 or a part thereof) of the CDRs, also preferably connected by a suitable framework sequence (s) or at least a part thereof. ) And at least some of the remaining two CDRs.

  According to another particularly preferred but non-limiting embodiment, such a portion or fragment comprises at least the CDR3, for example FR3, CDR3 and FR4 of the corresponding full-length Nanobody® of the invention (ie, for example, As described in International Application WO 03/050531 (Lasters et al.).

As already mentioned above, combining two or more such parts or fragments (ie from the same or separate amino acid sequences or Nanobodies® of the invention), ie the amino acid sequences of the invention, Nanobodies It is also possible to provide additional parts or fragments (as defined herein) of the ® or polypeptide. For example, it is also possible to combine one or more parts or fragments of an amino acid sequence, Nanobody® or polypeptide of the invention with one or more parts or fragments of a human _VH domain.

  According to one preferred embodiment, the part or fragment comprises one of the amino acid sequences SEQ ID NO: 60 to SEQ ID NO: 76, SEQ ID NO: 138 to SEQ ID NO: 141 and SEQ ID NO: 146 to SEQ ID NO: 157 or Nanobody®. Or at least 50%, preferably at least 60%, more preferably at least 70% of one of the polypeptides of SEQ ID NOs: 77 to 79, SEQ ID NOs: 142 to 145 and SEQ ID NOs: 158 to 165; Even more preferably, it has at least 80%, for example at least 90%, 95% or 99% or more sequence identity.

  The parts and fragments, and the nucleic acid sequences encoding them, are provided and can be combined in any manner known per se. For example, such a part or fragment can be obtained by inserting a stop codon into a nucleic acid encoding the full-length amino acid sequence, Nanobody® or polypeptide of the present invention, and then obtaining the nucleic acid thus obtained Can be obtained by expressing in a manner known per se (eg, as described herein). Alternatively, the nucleic acid encoding such a portion or fragment may appropriately limit the nucleic acid encoding the full-length amino acid sequence, Nanobody® or polypeptide of the invention, or may comprise such a nucleic acid. Can be obtained by a method known per se. Portions or fragments may also be prepared using peptide synthesis techniques known per se.

  The invention in its broadest sense also includes derivatives of the amino acid sequences, Nanobodies®, compounds or polypeptides of the invention. Such derivatives generally comprise one or more of the amino acid sequences, Nanobodies®, compounds or polypeptides of the invention, and / or one or more of the amino acid sequences, Nanobodies®, compounds or polypeptides of the invention. Can be obtained by modification of the amino acid residue, especially by chemical and / or biological (eg, enzymatic) modification.

  Examples of such modifications, as well as amino acid sequences, Nanobody® sequences, compounds or polypeptides that can be modified in such a form (ie, either the protein backbone or, preferably, the side chains) Examples of amino acid residues within the sequence, methods and techniques that can be used for the introduction of such modifications, and potential uses and advantages of such modifications will be apparent to those skilled in the art.

  For example, such modifications may add one or more functional groups, residues or moieties, particularly one or more desired properties or functionalities, to an amino acid sequence, Nanobody®, compound or polypeptide of the invention. The one or more functional groups, residues or moieties imparted can be incorporated into or on the amino acid sequence, Nanobody (R), compound or polypeptide of the invention (e.g., by a covalent bond or any other suitable (In form). Examples of such functional groups will be apparent to those skilled in the art.

  For example, such modifications increase the half-life, solubility and / or absorption of an amino acid sequence, Nanobody®, compound or polypeptide of the invention, that amino acid sequence, Nanobody ( (Registered trademark), reduce the immunogenicity and / or toxicity of the compound or polypeptide, eliminate or attenuate any undesired side effects of the amino acid sequences, Nanobodies (registered trademark), compounds or polypeptides of the present invention, and And / or confer other beneficial properties to the amino acid sequence, Nanobody (R), compound or polypeptide of the invention, and / or the unnecessary amino acid sequence, Nanobody (R), compound or polypeptide of the invention. One or more functional groups that reduce properties or provide any combination of two or more of the above (eg, Either by covalent bonding, or in any other suitable form) may include the introduction. Examples of such functional groups and techniques for introducing them will be apparent to those skilled in the art, and generally include all functional groups and techniques mentioned in the general background art cited above, as well as modification of pharmaceutical proteins, It may contain functional groups and techniques known per se, particularly for the modification of antibodies or antibody fragments (including ScFvs and single domain antibodies). See, for example, Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, PA (1980). As will also be apparent to those skilled in the art, such functional groups may be directly (eg, covalently) linked to, for example, an amino acid sequence, Nanobody®, compound or polypeptide of the invention, or may be optional. Can be linked via a suitable linker or spacer.

  One of the most widely used techniques to increase the half-life of a pharmaceutical protein and / or reduce its immunogenicity is to use a suitable pharmacologically acceptable polymer such as poly (ethylene glycol) (PEG) or a derivative thereof (eg, methoxy poly (ethylene glycol) or mPEG). In general, using any suitable form of pegylation, such as those used in the art for antibodies and antibody fragments, including but not limited to (single) domain antibodies and ScFvs Can be. For example, Chapman, Nat. Biotechnol., 54: 531-545 (2002), Veronese and Harris, Adv. Drug Deliv. Rev. 54: 453-456 (2003), Harris and Chess, Nat. Rev. Drug. Discov., 2: (2003) and WO 04/060965. A variety of reagents for protein pegylation are also commercially available, for example, from Nektar Therapeutics, USA.

  Preferably, site-specific pegylation is used, especially via cysteine residues (see for example Yang et al., Protein Engineering, 16, 10, 761-770 (2003)). For example, PEG may be attached to a naturally occurring cysteine residue in the amino acid sequence, Nanobody®, compound or polypeptide of the present invention, and the amino acid sequence, Nanobody®, compound or The polypeptide may be modified to appropriately introduce one or more cysteine residues that bind to PEG, or the amino acid sequence containing one or more cysteine residues that bind to PEG may be modified according to the invention. May be fused to the N-terminus and / or C-terminus of the Nanobody®, compound or polypeptide (all using protein engineering techniques known per se to those skilled in the art).

  Preferably, the molecular weight of the invention of the invention amino acid sequences, Nanobodies®, compounds or polypeptides is greater than 5000 (eg, greater than 10,000) and less than 200,000 (eg, less than 100,000). For example, PEG having a molecular weight in the range of 20,000 to 80,000 is used.

  Another, usually less preferred modification is a host cell used to express the amino acid sequence, Nanobody®, compound or polypeptide of the invention, usually as part of a translational and / or post-translational modification. And N-linked or O-linked glycosylation depending on

Still other modifications include one or more detectable labels or other signal-generating groups or moieties, depending on the use of the labeled amino acid sequence, Nanobody®, compound or polypeptide of the invention. May be included. Suitable labels and techniques for binding, using and detecting them will be apparent to those of skill in the art, and include, for example, fluorescent labels such as fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, And fluorescamine, and fluorescent metals such as ¹⁵² Eu, or other metals from the lanthanide species), phosphorescent labels, chemiluminescent labels or bioluminescent labels (eg luminal, isoluminal, theromatic acridinium ester) , Imidazole, acridinium salts, oxalates, dioxetane or GFP, and their analogs), radioisotopes (eg, ³ H, ¹²⁵ I, ³² P, ³⁵ S, ¹⁴ C, ⁵¹ Cr, ³⁶ Cl, ⁵⁷ Co, ^{58) Co,} 59 Fe And ⁷⁵ Se), metals, metal chelates or metallic cations (e.g. ^{99m Tc, 123 I, 111 In} , 131 I, 97 Ru, 67 Cu, 67 Ga, and ⁶⁸ Ga or the like of the metal cation), or in vivo, in vitro Or other metals or metal cations particularly suitable for in situ diagnostics and imaging, such as ( ¹⁵⁷ Gd, ⁵⁵ Mn, ¹⁶² Dy, ⁵² Cr, and ⁵⁶ Fe), and chromophores and enzymes (eg, malate dehydrogenase, staphylo) Coccus nuclease, δ-V-steroid isomerase, yeast alcohol dehydrogenase, α-glycerophosphate dehydrogenase, triosephosphate isomerase, biotin avidin peroxidase, horseradish peroxidase, alkaline phosphatase Fataze, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose -VI- phosphate dehydrogenase, glucoamylase, and acetylcholinesterase) include, but are not limited to. Other suitable labels will be apparent to those of skill in the art, and include moieties that can be detected using NMR or ESR spectroscopy.

  Such labeled amino acid sequences, Nanobodies®, compounds or polypeptides of the present invention may themselves (for example, depending on the choice of specific label, such as (ELISA, RIA, EIA and other “sandwich assays”). (Including known immunoassays) can be used for in vitro, in vivo or in situ assays, and for in vivo diagnostic and imaging purposes.

  As will be apparent to those skilled in the art, other modifications may include, for example, the introduction of a chelating group that chelates one of the metals or metal cations described above. For example, suitable chelating groups include, but are not limited to, diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

  Yet another modification may include the introduction of a functional group that is part of a specific binding pair, such as a biotin- (strept) avidin binding pair. Such a functional group may bind the amino acid sequence, Nanobody®, compound or polypeptide of the invention to another protein, polypeptide, which binds the other half of the binding pair (ie, through the formation of a binding pair). Or to a compound. For example, an amino acid sequence, Nanobody®, compound or polypeptide of the invention can be linked to another protein, polypeptide, compound or carrier that binds biotin and binds avidin or streptavidin. For example, such linked amino acid sequences, Nanobodies®, compounds or polypeptides of the invention can be used, for example, as reporters in diagnostic systems where a detectable signal generator binds to avidin or streptavidin. Such binding pairs can also be used, for example, to link the amino acid sequences, Nanobodies®, compounds or polypeptides of the invention to carriers, including carriers suitable for pharmaceutical purposes. One non-limiting example is the liposome formulation described by Cao and Suresh, Journal of Drug Targetting, 8, 4, 257 (2000). Such binding pairs can also be used to link a therapeutically active agent to the amino acid sequences, Nanobodies®, compounds or polypeptides of the invention.

  Other potential chemical and enzymatic modifications will be apparent to those skilled in the art. Such modifications may be introduced for research purposes (eg, to study function-activity relationships). See, for example, Lundblad and Bradshaw, Biotechnol. Appl. Biochem., 26, 143-151 (1997).

Preferably, the derivative is as defined herein (ie, as defined with respect to the amino acid sequence, Nanobody®, polypeptide, or compound itself) (as further described herein (actual or Affinity (preferably measured and / or expressed as an apparent) K _D value, a (real or apparent) K _A value, a k _on rate and / or a k _off rate, or alternatively as an IC ₅₀ value; It binds to protein F of hRSV. Also, such derivatives typically have hRSV neutralizing efficacy and / or efficacy as defined herein.

  As mentioned above, the present invention also relates to proteins or polypeptides consisting essentially of or comprising at least one amino acid sequence, Nanobody®, compound or polypeptide of the present invention. “Consisting essentially of” means that the amino acid sequence of any of the proteins or polypeptides of the invention is exactly the same as the amino acid sequence, Nanobody®, compound or polypeptide of the invention, or 1 to 20 amino acid residues, for example 1 to 10 amino acid residues, and preferably 1 to 6 amino acid residues (for example 1, 2, 3, 4, 5, or A limited number of amino acid residues (e.g., 6 amino acid residues) added to the amino acid sequence, Nanobody (R), compound or polypeptide at the amino terminus, carboxy terminus, or both the amino and carboxy termini. It means corresponding to the amino acid sequence, Nanobody®, compound or polypeptide of the invention.

The above amino acid residues may alter, modify or otherwise affect the (biological) properties of the amino acid sequence, Nanobody (R), compound or polypeptide of the present invention. May or may not provide, and may or may not add additional functionality to the amino acid sequence, Nanobody®, compound or polypeptide. For example, such an amino acid residue
a) For example, it may include an N-terminal Met residue resulting from expression in a heterologous host cell or organism.
b) During synthesis, a signal sequence or leader sequence may be formed that directs secretion of the amino acid sequence, Nanobody®, compound or polypeptide from the host cell. Suitable secretory leader peptides will be apparent to those of skill in the art, and may be as described further herein. Typically, such leader sequences are linked to the N-terminus of an amino acid sequence, Nanobody®, compound or polypeptide, but the invention is not limited in its broadest sense.
c) the amino acid sequence, Nanobody®, compound or polypeptide is directed to and / or penetrates or invades a particular organ, tissue, cell, or part or compartment of a cell And / or the amino acid sequence, Nanobody (R), compound or polypeptide is used in a cell membrane, a cell layer such as a layer of epithelial cells, a tumor, including a solid tumor, or a biological material such as the blood-brain barrier. Sequences or signals may be formed that allow the barrier to penetrate or traverse them. Examples of such amino acid sequences will be apparent to those skilled in the art. Some non-limiting examples are described in WO 03/026700, and in Temsamani et al., Expert Opin. Biol. Ther., 1, 773 (2001), Temsamani and Vidal, Drug Discov. Today, 9, 1012 (004) and the small peptide vector described in Rousselle, J. Pharmacol. Exp. Ther., 296, 124-131 (2001) ("Pep-trans vector"), and Zhao et al., Apoptosis, 8 , 631-637 (2003). C-terminal and N-terminal amino acid sequences for intracellular targeting of antibody fragments are described, for example, by Cardinale et al., Methods, 34, 171 (2004). Other suitable techniques for intracellular targeting include the expression and / or use of so-called “intracellular antibodies” comprising the amino acid sequences, Nanobodies®, compounds or polypeptides of the invention, as described below. Accompanied by
d) “Tags”, eg, amino acids that allow or facilitate the purification of amino acid sequences, Nanobodies®, compounds or polypeptides using, for example, affinity techniques directed to the above sequences or residues. Sequences or residues may be formed. Thereafter, the above sequences or residues may be removed (eg, by chemical or enzymatic cleavage) to yield an amino acid sequence, Nanobody®, compound or polypeptide sequence (for this purpose, the tag May optionally be linked to an amino acid sequence, Nanobody®, compound or polypeptide via a cleavable linker sequence, or may contain a cleavable motif). Some preferred but non-limiting examples of such residues are multiple histidine residues, glutathione residues, and myc tags such as AAAEQKLISEEDLNGAA (SEQ ID NO: 111).
e) It can be one or more amino acid residues that can be functionalized and / or function as a binding site for a functional group. Suitable amino acid residues and functional groups will be apparent to those skilled in the art, and the amino acid residues and functional groups referred to herein for the amino acid sequences, Nanobodies®, compounds or polypeptide derivatives of the present invention may be modified. But not limited thereto.

  The invention further relates to methods of preparing the amino acid sequences, Nanobodies®, polypeptides, compounds, nucleic acids, host cells, products and compositions described herein.

  The amino acid sequences, Nanobodies®, polypeptides and nucleic acids of the invention can be prepared in a manner known per se, as will be clear to the skilled person from the further description herein. For example, the amino acid sequences, Nanobodies®, and polypeptides of the invention may be used for the preparation of antibodies, particularly for the preparation of antibody fragments, including but not limited to (single) domain antibodies and ScFv fragments. It can be prepared by any method known per se. Some preferred but non-limiting methods for preparing amino acid sequences, Nanobodies®, polypeptides and nucleic acids include the methods and techniques described herein.

A method for preparing an amino acid sequence of the invention, a Nanobody® of the invention, a polypeptide of the invention, or a monovalent construct of the invention comprises:
In a suitable host cell or host organism (also referred to herein as a "host of the invention") or a nucleic acid encoding an amino acid sequence, Nanobody® or polypeptide of the invention as described above (as described herein). Expressing the "nucleic acid of the invention" in another suitable expression system, optionally followed by:
Isolating and / or purifying the amino acid sequence, Nanobody (registered trademark) or polypeptide of the present invention thus obtained.

In particular, such a method
Culturing and / or maintaining the host of the invention under conditions such that the host of the invention described above expresses and / or produces at least one amino acid sequence, Nanobody® and / or polypeptide of the invention. Process and, optionally,
Isolating and / or purifying the thus obtained amino acid sequence, Nanobody (registered trademark) or polypeptide of the present invention.

  Accordingly, the present invention relates to nucleic acids or nucleotide sequences encoding the amino acid sequences, Nanobodies®, polypeptides or monovalent constructs of the invention (also referred to as “nucleic acid sequences of the invention” or “nucleotide sequences of the invention”). ). The nucleic acid of the present invention can be in the form of single-stranded or double-stranded DNA or RNA, and preferably in the form of double-stranded DNA. For example, the nucleotide sequence of the present invention may be genomic DNA, cDNA or synthetic DNA (such as DNA having codon usage particularly adapted for expression in the host cell or host organism of interest).

  According to one embodiment of the invention, the nucleic acids according to the invention are in an essentially isolated form, as defined herein. The nucleic acids of the invention may be in the form of a vector, such as a plasmid, cosmid or YAC, present in the vector, and / or may be a part thereof, also in an essentially isolated form. Good.

  The nucleic acids of the invention can be prepared or obtained in a manner known per se, based on the amino acid sequences, Nanobodies® and / or polypeptides of the invention described herein. And / or from suitable natural sources. Also, as will be apparent to one of skill in the art, to prepare a nucleic acid of the invention, for example, an amino acid sequence or at least one nucleotide sequence encoding Nanobody®, and eg, encoding one or more linkers Several nucleotide sequences, such as nucleic acids, can be linked together in any suitable manner.

  Techniques for producing the nucleic acids of the invention will be apparent to those skilled in the art, for example, automated DNA synthesis, site-directed mutagenesis, two or more native and / or synthetic sequences (or two or more thereof). ), The introduction of mutations to express the truncated expression product (eg, to produce cassettes and / or regions that can be easily digested and / or ligated using suitable restriction enzymes). This includes, but is not limited to, the introduction of one or more restriction sites and / or the introduction of mutations by a PCR reaction using one or more “mismatched” primers. These and other techniques will be apparent to those of ordinary skill in the art, and also refer to standard handbooks, such as the Sambrook et al. And Ausubel et al. References cited above, and the following examples.

  The nucleic acids of the invention may also take the form of, be present in, and / or be part of, a genetic construct, as will be apparent to those of skill in the art. Such genetic constructs are generally referred to, for example, by one or more suitable regulatory elements (eg, suitable promoter (s), enhancer (s), terminator (s), etc.), and as referred to herein. It comprises at least one nucleic acid of the invention optionally linked to one or more elements of a genetic construct known per se, such as a further element of a given genetic construct. Such a genetic construct comprising at least one nucleic acid of the invention is also referred to herein as a "gene construct of the invention".

  The gene construct of the present invention may be DNA or RNA, and is preferably double-stranded DNA. In addition, the gene construct of the present invention may be in a form suitable for transformation of a target host cell or host organism, a form suitable for integration into genomic DNA of a target host cell, or an independent form in a target host organism. It may take any form suitable for replication, maintenance and / or inheritance. For example, the gene construct of the present invention can be in the form of a vector such as a plasmid, cosmid, YAC, viral vector or transposon. In particular, the vector may be an expression vector, ie a vector capable of providing expression in vitro and / or in vivo (eg in a suitable host cell, host organism and / or expression system).

In one preferred, but non-limiting embodiment, the genetic construct of the invention comprises:
a) at least one nucleic acid of the invention operably linked to b);
b) comprising one or more regulatory elements such as a promoter, and optionally a suitable terminator, and optionally c) one or more further elements of a genetic construct known per se, wherein " The terms “regulatory element”, “promoter”, “terminator”, and “operably linked” have their ordinary meaning in the art (as further described herein) and Said "additional elements" present in the construct include, for example, 3′- or 5′-UTR sequences, leader sequences, selectable markers, expression markers / reporter genes, and / or enhance transformation (integration) efficiency. Or it may be an element that can be increased. These and other elements suitable for such a genetic construct will be apparent to those skilled in the art, such as the type of construct used, the host cell or host organism of interest, the method of expressing the nucleotide sequence of the invention of interest (eg, (Through constitutive expression, transient expression, inducible expression, etc.), and / or the transformation method used. The regulatory sequences, promoters and terminators known per se, for example for the expression and production of antibodies and antibody fragments (including but not limited to (single) domain antibodies and ScFv fragments) are essentially It may be used in a similar manner.

  Preferably, in the genetic construct of the invention, at least one of the above-mentioned nucleic acids of the invention, and the above-mentioned regulatory elements, and optionally one or more further elements, are "operably linked" to each other. , Which usually means that they are in a functional relationship with each other. For example, if the promoter is capable of initiating or otherwise controlling / regulating the transcription and / or expression of the coding sequence (where the coding sequence is "under control" of the promoter). , Is operably linked to the coding sequence. Generally, when two nucleotide sequences are operably linked, they have the same orientation and are usually in the same reading frame. Usually, these sequences are essentially contiguous, but this is not required.

  Preferably, the regulatory and further elements of the genetic construct of the invention are such that they can confer the biological function of interest in the host cell or host organism of interest.

  For example, a promoter, enhancer or terminator shall be “operable” in a host cell or host organism of interest, which (for example) means that the promoter is operable (as defined herein). It means that the transcription and / or expression of the linked nucleotide sequence (eg, coding sequence) can be controlled / regulated by initiation or other methods.

  Some particularly preferred promoters include promoters known per se for expression in the host cells referred to herein, especially those referred to herein and / or used in the Examples. Promoters for expression in bacterial cells such as, but not limited to.

  The selectable marker can be used to identify a host cell and / or host organism that has (successfully) transformed with the nucleotide sequence of the present invention, and a host cell and / or host organism that has not (successfully) transformed (ie, Under good selection conditions). Some preferred, but non-limiting examples of such markers include genes that confer resistance to an antibiotic (eg, kanamycin or ampicillin), genes that confer temperature resistance, or cells that are not transformed or survive. Genes that maintain a host cell or organism in the absence of certain factors, compounds and / or (food) components in the culture medium.

  The leader sequence shall be such that it allows for the desired post-translational modification (in the host cell or host organism of interest) and / or directs the transcribed mRNA to the desired part or organelle of the cell. . The leader sequence may also secrete the expression product from the cells. As such, the leader sequence may be any prosequence, presequence, or preprosequence operable in the host cell or host organism. Leader sequences may not be required for expression in bacterial cells. For example, leader sequences known per se for the expression and production of antibodies and antibody fragments, including but not limited to single domain antibodies and ScFv fragments, are used in an essentially similar manner. be able to.

  The expression marker or reporter gene is such that (in the host cell or host organism) the expression of the gene construct (the gene or nucleotide sequence present in) can be detected. The expression marker may optionally include an expression product, such as a particular portion or organelle of a cell, and / or a particular cell (s), tissue (s), organ (s), or portion (s) of a multicellular organism. Yes). Such a reporter gene may be expressed as a protein fusion with the amino acid sequence, Nanobody® or polypeptide of the invention. Some preferred but non-limiting examples include fluorescent proteins such as GFP.

  Some preferred but non-limiting examples of suitable promoters, terminators, and additional elements include those that can be used for expression in the host cells referred to herein, and those specifically described herein. Those mentioned herein and / or those suitable for expression in bacterial cells, such as those used in the examples below. Some (additional) of promoters, selectable markers, leader sequences, expression markers and additional elements (eg, terminators, transcription and / or translation enhancers, and / or integration factors) that may be present / used in the genetic constructs of the present invention. For non-limiting examples, see Handbooks reviewed by Sambrook et al. And Ausubel et al., Supra, and WO 95/07463, WO 96/23810, WO 95/95. No. 07463, WO 95/21191, WO 97/11094, WO 97/42320, WO 98/06737, WO 98/21355, U.S. Pat. No. 7,207, Reference is made to the examples shown in U.S. Pat. No. 410, U.S. Pat. No. 5,693,492 and EP 1085089. Other examples will be apparent to those skilled in the art. Reference is also made to the general background references cited above and the further references cited herein.

  Genetic constructs of the invention are typically prepared using one or more of the nucleotide sequences of the invention using one or more of the above-described techniques, for example, using the techniques described in the Handbook outlined by Sambrook et al. And Ausubel et al. Can be obtained by appropriate connection with additional elements of

  In many cases, the genetic constructs of the invention are obtained by inserting the nucleotide sequence of the invention into a suitable (expression) vector known per se. Some preferred but non-limiting examples of suitable expression vectors are those used in the Examples below, and those mentioned herein.

The nucleic acids of the invention and / or the genetic constructs of the invention are used for transforming host cells or organisms, ie for expressing and / or producing the amino acid sequences, Nanobodies® or polypeptides of the invention. can do. Suitable hosts or host cells will be apparent to those skilled in the art; for example, any suitable fungal, prokaryotic, or eukaryotic cell or cell line or any suitable fungal, prokaryotic, or eukaryotic, such as a bacterial strain ( Gram-negative strains such as Escherichia coli strains, strains of the genus Proteus such as Proteusmirabilis, strains of the genus Pseudomonas such as Pseudomonas fluorescens; and Bacillus subtilis or Bacillus brevis Of the genus Bacillus, strains of the genus Streptomyces such as Streptomyces lividans, strains of staphylococci such as Staphylococcus carnosus, and lactococci such as Lactococcus lactis Gram-positive strains such as, but not limited to)
Fungal cells (Trichoderma genus such as Trichoderma reesei (Trichodermareesei), Neurospora genus such as Neurospora crassa, Sordaria genus such as Sordaria macrospora, Aspergillus niger (Aspergillusniger) Such as, but not limited to, cells from the genus Aspergillus or other filamentous fungi),
Yeast cells (genus Saccharomyces such as Saccharomyces cerevisiae), genus Schizosaccharomyces such as Schizosaccharomyces pombe, genus Pichia pastoris or genus Pichia methanolica such as Pichia methanolica (Pichia methanolica) Hansenula genus such as polymorpha (Hansenula polymorpha), Kluyveromyces lactis and the like genus Kluyveromyces, Arksula adeninivorans (Arxula adeninivorans) and the like, Yarrowia lipolytica (Yarrowialipolytica) and other genus Yarrowialipolytica But not limited to these),
Amphibian cells or cell lines, such as Xenopus oocytes,
Insect-derived cells or cell lines, for example, cells / cell lines derived from Lepidoptera, including, but not limited to, Sci-fi armyworm SF9 cells and Sf21 cells, or cells / cells derived from Drosophila, such as Schneider cells and Kc cells. stock,
Plants or plant cells, eg, tobacco plants, and / or mammalian cells or cell lines, eg, CHO cells, BHK cells (eg, BHK-21 cells), and HeLa cells, COS cells (eg, COS-7 cells), and PER . Human-derived cells or cell lines, such as, but not limited to, C6 cells, cells or cell lines derived from humans, cells or cell lines derived from mammals, and antibodies and antibody fragments ((single) domain antibodies and All other hosts or host cells known per se for the expression and production of (including but not limited to ScFv fragments) may be apparent to those skilled in the art. The general background art cited above and, for example, WO 94/29457, WO 96/34103, WO 99/42077, Frenken et al., (1998) supra, Riechmann and Muyldermans, (1999) ibid, van der Linden, (2000) ibid, Thomassen et al., (2002) ibid, Josten et al., (2003) ibid, Josten et al., (2005) ibid, and herein. Reference is made to the further references cited.

  The amino acid sequences, Nanobodies® and polypeptides of the invention can be introduced into one or more cells, tissues or organs of a multicellular organism, eg, for prophylactic and / or therapeutic purposes (eg, gene therapy). Can also be expressed. To this end, the nucleotide sequence of the invention may be derived in any suitable manner, for example neat (eg using liposomes) or from a suitable (eg retrovirus such as adenovirus, parvovirus such as adeno-associated virus) After insertion into a gene therapy vector, it can be introduced into cells or tissues. As will be apparent to those skilled in the art, such gene therapy involves transferring a nucleic acid of the invention or a suitable gene therapy vector encoding a nucleic acid of the invention to a patient or a particular cell or a particular tissue or organ of the patient. The administration can be performed in vivo and / or in situ in the body of the patient, or can be performed in a suitable (often explanted lymphocyte, bone marrow aspirate or tissue biopsy sample, etc.) The cells (collected from the patient's body) can be suitably (re) introduced into the patient's body after being treated in vitro with the nucleotide sequence of the present invention. All of these are described, for example, in Culver, KW, "Gene Therapy", 1994, p. Xii, Mary Ann Liebert, Inc., Publishers, New York, NY, Giordano, Nature F Medicine 2: (1996), 534-539, Schaper, Res. 79 (1996), 911-919, Anderson, Science 256 (1992), 808-813, Verma, Nature 389 (1994), 239, Isner, Lancet 348 (1996), 370-374, Muhlhauser, Circ. Res. 77 (1995), 1077-1086, Onodera, Blood 91: (1998), 30-36, Verma, Gene Ther. 5 (1998), 692-699, Nabel, Ann. NY Acad. Sci .: 811. (1997), 289-292, Verzeletti, Hum. Gene Ther. 9 (1998), 2243-51, Wang, Nature Medicine 2 (1996), 714-716, International Publication No. 94/29469, International Publication No. 97 /. 00957, US Patent No. 5,580,859, US Patent No. 5,589,466 or Schaper, Current Opinion in Biotechnology 7 (1996), 635-640. And can be done using a delivery systemFor example, in situ expression of ScFv fragments (Afanasieva et al., Gene Ther., 10, 1850-1859 (2003)) and diabodies (Blanco et al., J. Immunol., 171, 1070-1077 (2003)) It is described in the art.

  For the expression of amino acid sequences, Nanobodies® or polypeptides in cells, these are, for example, WO 94/02610, WO 95/22618, US Pat. 03/014960, Cattaneo, A. & Biocca, S. (1997) Intracellular Antibodies: Development and Applications. Landes and Springer-Verlag, and Kontermann, Methods 34, (2004), 163-170). Can be expressed as "internal antibody".

  General techniques for introducing transgenes into mammals include the amino acid sequences, Nanobodies®, and polypeptides of the invention in the milk of a transgenic mammal, such as, for example, rabbit, cow, goat, or sheep milk. See, for example, U.S. Patent Nos. 6,741,957, 6,304,489, and 6,849,992), plants, or leaves, flowers, fruits, seeds, roots. Can also be produced in some parts of the plant, including but not limited to tubers (eg, tobacco, corn, soy, or alfalfa), or, for example, in silkworm pupae.

  Furthermore, the amino acid sequences, Nanobodies® and polypeptides of the present invention may be expressed and / or produced in cell-free expression systems, and suitable examples of such systems will be apparent to those skilled in the art. Would. Some preferred but non-limiting examples include expression in a wheat malt system, rabbit reticulocyte lysate, or a system using E. coli by the method of Zubay.

  As mentioned above, one of the advantages of using Nanobodies® is that polypeptides based thereon can be prepared by expression in a suitable bacterial system, and suitable bacterial expression systems, vectors, Host cells, regulatory elements and the like will be apparent to those skilled in the art, for example, from the references described above. However, it should be noted that the invention is not limited in its broadest sense to expression in bacterial systems.

  Preferably, the present invention employs an expression system (in vivo or in vitro), such as a bacterial expression system, which provides the polypeptide of the invention in a form suitable for pharmaceutical use, but such expression systems are also known to those of skill in the art. It will be obvious. As will be apparent to one of skill in the art, polypeptides of the invention suitable for pharmaceutical use can also be prepared using peptide synthesis methods.

  In industrial-scale production, preferred heterologous hosts for (industrial) production of Nanobody® or Nanobody®-containing protein therapeutics include large-scale expression / production / fermentation, especially large-scale production. And Escherichia coli, Pichia pastoris, and budding yeast strains suitable for expression / production / fermentation in pharmaceutical applications. Suitable examples of such strains will be clear to the skilled person. Such strains and production / expression systems are also available from companies such as Biovitrum (Uppsala, Sweden).

  Alternatively, mammalian cell lines, particularly Chinese hamster ovary (CHO) cells, can be used for large-scale expression / production / fermentation, particularly for large-scale pharmaceutical applications. Such expression / production systems are also available from some of the companies mentioned above.

  The choice of a particular expression system will depend in part on the particular post-translational modification, more specifically the requirements for glycosylation. The production of recombinant proteins containing Nanobodies® where glycosylation is desired or required requires the use of a mammalian expression host capable of glycosylating the expressed protein. In this context, it will be apparent to one of skill in the art that the resulting glycosylation pattern (ie, the type, number and location of binding residues) will depend on the cell or cell line used for expression. Preferably, a human (ie, which gives a protein having an essentially human glycosylation pattern) cells or cell lines, or essentially and / or functionally identical to human glycosylation, or at least human Another mammalian cell line that can provide a glycosylation pattern that mimics glycosylation is used. In general, prokaryotic hosts such as E. coli do not have the ability to glycosylate proteins, and the use of lower eukaryotic cells, such as yeast, usually results in a different glycosylation pattern than human glycosylation. Nevertheless, it is to be understood that all of the above host cells and expression systems can be used in the present invention, depending on the desired amino acid sequence, Nanobody (R) or polypeptide to be obtained.

  Thus, according to one non-limiting embodiment of the invention, the amino acid sequence, Nanobody® or polypeptide of the invention is glycosylated. According to another non-limiting embodiment of the present invention, the amino acid sequence, Nanobody® or polypeptide of the present invention is not glycosylated.

  According to one preferred but non-limiting embodiment of the invention, the amino acid sequence, Nanobody® or polypeptide of the invention is a bacterial cell, in particular a bacterial cell suitable for large-scale pharmaceutical production, For example, it is produced in cells of the above strain.

  According to another preferred but non-limiting embodiment of the invention, the amino acid sequence, Nanobody® or polypeptide of the invention is a yeast cell, in particular a yeast cell suitable for large-scale pharmaceutical production. For example, in cells of the species described above.

  According to yet another preferred but non-limiting embodiment of the invention, the amino acid sequence, Nanobody® or polypeptide of the invention is a mammalian cell, in particular a human cell or human cell line. It is produced in cells, more specifically in human cells or human cell lines suitable for large-scale pharmaceutical production, such as those of the above cell lines.

  When expression in a host cell is used to produce the amino acid sequences, Nanobodies® and polypeptides of the present invention, the amino acid sequences, Nanobodies® and polypeptides of the present invention may be intracellularly (eg, cytoplasmic). Medium, in the periplasm, or in inclusion bodies) and then isolated from the host cells, optionally further purified, or produced extracellularly (eg, in the medium in which the host cells are cultured) and then from the medium. It may be isolated and optionally further purified. When using eukaryotic host cells, the resulting amino acid sequences, Nanobodies®, polypeptides and proteins, are usually much more easily isolated and processed downstream, and are usually produced extracellularly. Preferably. Usually, bacterial cells, such as the E. coli strains mentioned above, do not secrete proteins extracellularly, except for some proteins, such as toxins and blood poisons, and produce secretions in Escherichia coli, To transfer proteins to the periplasmic space through Production in the periplasm has several advantages over production in the cytoplasm. For example, the N-terminal amino acid sequence of the secretory product may be identical to the native gene product after cleavage of the secretory signal sequence by a specific signal peptidase. Also, protease activity appears to be much lower in the periplasm than in the cytoplasm. Further, in the periplasm, the amount of contaminating proteins is small, so that protein purification is easier. Another advantage is that disulfide bonds can be formed in the correct position because the periplasm provides a more oxidative environment than the cytoplasm. Proteins that are overexpressed in E. coli are often found in insoluble aggregates called inclusion bodies. These inclusions can be present either in the cytoplasm or in the periplasm, and the recovery of biologically active proteins from these inclusions requires a denaturation / refolding process. Many recombinant proteins, including therapeutic proteins, are recovered from inclusion bodies. Alternatively, as will be apparent to one of skill in the art, recombinant strains of bacteria that have been genetically modified to secrete the desired protein, particularly the amino acid sequences, Nanobodies® or polypeptides of the invention, may be prepared. Can be used.

  Thus, according to one non-limiting embodiment of the invention, the amino acid sequences, Nanobodies® or polypeptides of the invention are produced intracellularly and from host cells, in particular in bacterial cells or in bacterial cells. Amino acid sequence, Nanobody® or polypeptide isolated from inclusion bodies. According to another non-limiting embodiment of the present invention, the amino acid sequence, Nanobody® or polypeptide of the present invention is produced extracellularly and isolated from the medium in which the host cell is cultured. , Nanobodies® or polypeptides.

Some preferred but non-limiting promoters for use with these host cells include:
For expression in E. coli, the lac promoter (and their derivatives such as the lacUV5 promoter), the arabinose promoter, the left (PL) and right (PR) promoters of λ phage, the promoter of the trp operon, the hybrid lac / trp promoter (tac and trc), the T7-promoter (more specifically the promoter of the T7-phage gene 10), and other T-phage promoters, the promoter of the Tn10 tetracycline resistance gene, one or more copies of the foreign control operator sequence A recombinant mutant of the promoter;
ADH1 (alcohol dehydrogenase 1), ENO (enolase), CYC1 (cytochrome iso-1), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), PGK1 (phosphoglycerin) for expression in budding yeast Constitutive promoters of acid kinase) and PYK1 (pyruvate kinase); regulated promoters of GAL1, GAL10, GAL7 (galactose metabolizing enzyme), ADH2 (alcohol dehydrogenase 2), PHO5 (acid phosphatase), and CUP1 (copper metallothionein) A foreign promoter of CaMV (Cauliflower Mosaic Virus 35S promoter);
AOX1 promoter (alcohol oxidase I) for expression in Pichia pastoris;
Human cytomegalovirus (hCMV) immediate early enhancer / promoter for expression in mammalian cells, human cytomegalovirus (hCMV) containing two tetracycline operator sequences such that the promoter can be controlled by a Tet repressor Early promoter mutant, herpes simplex virus thymidine kinase (TK) promoter, rous sarcoma virus long terminal repeat (RSV LTR) enhancer / promoter, elongation factor 1α (hEF-1α) promoter from human, chimpanzee, mouse, or rat , SV40 early promoter, HIV-1 long terminal repeat promoter, and β-actin promoter.

Some preferred but non-limiting vectors for use with these host cells include:
Vectors for expression in mammalian cells: pMAMneo (Clontech), pcDNA3 (Invitrogen), pMC1neo (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593), pBPV-1 (8-2) (ATCC) 37110), pdBPV-MMTneo (342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460) and 1ZD35 (ATCC 37) Virus-based expression systems, such as those based on viruses;
Vectors for expression in bacterial cells: pET vector (Novagen) and pQE vector (Qiagen);
Vectors for expression in yeast or another fungal cell: pYES2 (Invitrogen) and Pichia expression vector (Invitrogen);
Vectors for expression in insect cells: pBlueBacII (Invitrogen) and other baculovirus vectors;
Vectors for expression in plants or plant cells: include, for example, cauliflower mosaic virus or tobacco mosaic virus, vectors based on suitable strains of Agrobacterium, or Ti-plasmid based vectors.

Some preferred but non-limiting secretory sequences for use with these host cells include:
For use in bacterial cells such as E. coli: PelB, Bla, OmpA, OmpC, OmpF, OmpT, StII, PhoA, PhoE, MalE, Lpp, LambB, etc .; TAT signal peptide, hemolysin C-terminal secretion signal;
For use in yeast: α-mating factor prepro sequence, phosphatase (pho1), invertase (Suc), etc .;
For use in mammalian cells: specific signals (if the target protein is of eukaryotic origin), including the mouse Igκ chain V-J2-C signal peptide.

  Suitable techniques for transforming a host or host cell of the invention will be apparent to those skilled in the art and may depend on the host cell / host organism of interest and the genetic construct used. Similarly, reference is made to the above handbooks and patent applications.

  After transformation, steps can be performed to detect and select for host cells or organisms that have been successfully transformed with the nucleotide sequence / gene constructs of the present invention. This step may be, for example, a step of selecting based on a selectable marker present in the gene construct of the present invention, or a step comprising detecting the amino acid sequence of the present invention using, for example, a specific antibody.

  A transformed host cell (which can take the form of a stable cell line) or host organism (which can take the form of a stable mutant or strain) is a further aspect of the invention. Eggplant

  Preferably, these host cells or organisms (in the case of host organisms, in at least one cell, part, tissue or organ thereof) express an amino acid sequence, Nanobody® or polypeptide of the invention. Or (at least) (eg, under suitable conditions). The present invention also includes further generations, progeny and / or progeny of the host cells or host organisms of the present invention, which may be obtained, for example, by cell division or sexual or asexual reproduction.

  In order to express / obtain the amino acid sequence, Nanobody® or polypeptide of the invention, the transformed host cell or transformed host organism generally comprises a (desired) amino acid sequence of the invention. , Nanobody® or polypeptides can be maintained, maintained and / or cultured under conditions such that they are expressed / produced. Suitable conditions will be clear to the skilled person and usually depend on the host cell / host organism used and on the regulators controlling the expression of the (related) nucleotide sequences of the invention. Here again, reference is made to the handbooks and patent applications mentioned in the above paragraph regarding the gene constructs of the invention.

  In general, suitable conditions include the use of a suitable medium, the presence of a suitable diet and / or a suitable nutrient, the use of a suitable temperature, and the presence of any suitable inducer or compound (eg, a nucleotide of the invention). (If the sequence is under the control of an inducible promoter), all of which can be selected by one skilled in the art. Again, under such conditions, the amino acid sequences of the present invention can be expressed continuously, transiently, or only when appropriately induced.

  The amino acid sequences, Nanobodies® or polypeptides of the present invention may be (first) produced in immature form (described above) and then post-translationally modified depending on the host cell / host organism used. Will be apparent to those skilled in the art. Also, the amino acid sequence, Nanobody® or polypeptide of the invention may be glycosylated, again depending on the host cell / host organism used.

  The amino acid sequence, Nanobody® or polypeptide of the invention may then be obtained from a host cell / host organism and / or from the medium in which said host cell or host organism has been cultured, by (preparative) chromatographic methods, and / or Electrophoresis, fractional precipitation, affinity (eg, using a specific cleavable amino acid sequence fused to an amino acid sequence of the invention, Nanobody® or polypeptide), and / or preparative immunization (ie, (Using an antibody against the amino acid sequence to be isolated) and the like, using known protein isolation and / or purification techniques.

  The invention further relates to at least one amino acid sequence of the invention (or a suitable fragment thereof), at least one Nanobody of the invention, at least one polypeptide of the invention, at least one compound of the invention or The construct, at least one monovalent construct of the invention, and / or at least one nucleic acid of the invention, and optionally one or more of such compositions known per se, depending on the intended use of the composition Products or compositions containing or containing further components. Such a product or composition may be, for example, a pharmaceutical composition (as described herein), a veterinary composition or product (also described herein), or a diagnostic composition. The composition may be: Some preferred, but non-limiting examples of such products or compositions will become clear from the further description herein.

  In general, for pharmaceutical use, the amino acid sequences, Nanobodies® and polypeptides of the invention are combined with at least one amino acid sequence, Nanobody® or polypeptide of the invention, and at least one pharmaceutically acceptable Pharmaceutical preparation or composition comprising a possible carrier, diluent or excipient and / or adjuvant, and optionally one or more further pharmacologically active polypeptides and / or compounds Can be formulated as By way of non-limiting example, such formulations may be for oral administration, for parenteral administration (such as by intravenous, intramuscular or subcutaneous injection, or intravenous infusion), for topical administration, inhalation, transdermal It can take any form suitable for administration by patches, implants, suppositories and the like. Such suitable dosage forms may be solid, semi-solid or liquid, depending on the method of administration, and the method and carrier to be used in the preparation, will be apparent to those skilled in the art, and are herein defined. Further explanation will be given.

  Thus, in a further aspect, the invention relates to at least one amino acid sequence according to the invention, at least one Nanobody® according to the invention, at least one compound or construct according to the invention or at least one polymorph according to the invention. It relates to a pharmaceutical composition comprising the peptide, at least one suitable (ie suitable for pharmaceutical use) carrier, diluent or excipient, and optionally one or more further active substances.

In general, the amino acid sequences, Nanobodies®, compounds, constructs and polypeptides of the invention can be formulated and administered by any suitable method known per se, for example, as described above. General background arts (in particular, WO 04/041862, WO 04/041863, WO 04/041865, WO 04/041867 and WO 08/020079), and ^{Remington's Pharmaceutical Sciences, 18 th Ed} ., MackPublishing Company, USA (1990), Remington, the Science and Practice of Pharmacy, 21st Edition, Lippincott Williams andWilkins (2005) standard handbooks of literature, such as, or TherapeuticAntibodies (S. Dubel, Ed.), Wiley, Weinhelm, 2007 Handbook (see, for example, pages 252 to 255).

  For example, the amino acid sequences, Nanobodies®, compounds, constructs and polypeptides of the invention may be used for conventional antibodies and antibody fragments (including ScFv and diabodies) and other pharmaceutically active proteins. It can be formulated and administered in any manner known per se. Such formulations and methods of preparing them will be apparent to those skilled in the art, for example, parenteral (eg, intravenous, intraperitoneal, subcutaneous, intramuscular, intraluminal, intraarterial or intrathecal) or topical (ie, Preparations suitable for transdermal or intradermal) administration are mentioned.

  Preparations for parenteral administration may be, for example, sterile solutions, suspensions, dispersions or emulsifiers suitable for infusion or injection. Suitable carriers or diluents for such preparations include, for example, and without limitation, those mentioned on page 143 of WO 08/020079. Usually, aqueous solutions or suspensions are preferred.

  The amino acid sequences, Nanobodies®, compounds, constructs and polypeptides of the invention can also be administered using gene therapy delivery methods. See, for example, U.S. Patent No. 5,399,346, which is incorporated by reference in its entirety. Using a gene therapy delivery method, primary cells transfected with a gene encoding an amino acid sequence, Nanobody® or polypeptide of the invention can be used to remove specific organs, tissues, grafts, tumors or cells. It can be further transfected with a tissue-specific promoter for targeting, and further transfected with signal and stabilizing sequences for expression localized within the cell.

  Thus, the amino acid sequences, Nanobodies®, compounds, constructs and polypeptides of the invention can be combined with a pharmaceutically acceptable vehicle, such as an inert diluent or an assimilable edible carrier, for example, orally. Can be systemically administered. They may be enclosed in hard or soft shell gelatin capsules, may be compressed as tablets, or may be incorporated directly with the food in the patient's diet. For oral therapeutic administration, the amino acid sequences, Nanobodies®, compounds, constructs and polypeptides of the present invention may be combined with one or more excipients, ingestible tablets, buccal tablets, troches, and capsules. , Elixirs, suspensions, syrups, wafers and the like. Such compositions and preparations should contain at least 0.1% of an amino acid sequence, Nanobody®, compound, construct or polypeptide of the invention. Their proportion in the compositions and preparations may, of course, be varied and may conveniently be between about 2% to about 60% of the weight of a given unit dosage form. The amount of the amino acid sequence, Nanobody®, compound, construct or polypeptide of the invention in such therapeutically useful compositions is such that an effective dosage level is obtained.

  Tablets, troches, pills, capsules and the like also refer to binders, excipients, disintegrants, lubricants, sweeteners or flavors, for example, pages 143 to 144 of WO 08/020079. May be contained. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For example, tablets, pills, or capsules may be coated with gelatin, wax, shellac, sugar or the like. Syrups or elixirs may contain the amino acid sequences, Nanobodies®, compounds, constructs and polypeptides of the present invention, sucrose or fructose as a sweetening agent, methyl or propyl parabens as preservatives, a dye and cherry or orange flavor. It may contain a fragrance. Of course, all materials used in preparing any unit dosage form must be pharmaceutically acceptable and substantially non-toxic in the amounts employed. Further, the amino acid sequences, Nanobodies®, compounds, constructs and polypeptides of the invention may be incorporated into sustained-release preparations and devices.

  Preparations and formulations for oral administration may also be provided with an enteric coating, which allows the constructs of the present invention to be resistant to the gastric environment and enter the intestine. More generally, preparations and formulations for oral administration can be suitably formulated for delivery to any desired part of the gastrointestinal tract. Also, suitable suppositories may be used for delivery to the gastrointestinal tract.

  The amino acid sequences, Nanobodies®, compounds, constructs and polypeptides of the present invention may also be administered intravenously or intraperitoneally by infusion or injection, as further described in WO 08/020079 at pages 144 and 145. It may be administered internally.

  For topical administration, the amino acid sequences, Nanobodies®, compounds, constructs and polypeptides of the invention can be applied in pure form, ie when in liquid form. However, in general, they will further comprise a composition or formulation together with a dermatologically acceptable carrier, which may be solid or liquid, as further described on page 145 of WO 08/020079. Is desirably administered to the skin.

  Generally, the concentration of the amino acid sequences, Nanobodies®, compounds, constructs and polypeptides of the invention in a liquid composition such as a lotion will be about 0.1 wt% to 25 wt%, preferably about 0.5 wt% to 10 wt%. The concentration in a semi-solid or solid composition such as a gel or powder is about 0.1 wt% to 5 wt%, preferably about 0.5 wt% to 2.5 wt%.

  In a preferred embodiment, the amino acid sequences, Nanobodies®, compounds, constructs, and / or polypeptides of the invention and / or compositions comprising the same are administered to pulmonary tissue. In the context of the present invention, "lung tissue" is similar to lung tissue or lung for the purposes of the present invention. The lung contains two distinct areas, the airway area and the respiratory area, within which there are airway and vascular compartments (eg "Pulmonary Drug Delivery" edited by Karoline Bechtold-Peters and Henrik Luessen, 2007, ISBN 978-3. -87193-322-6pages 16-28).

  For pulmonary delivery, the amino acid sequences, Nanobodies®, compounds, constructs and polypeptides of the invention can be applied in pure form, ie, when in liquid or dry powder. However, it is preferred to deliver them to lung tissue as a composition or formulation comprising the amino acid sequences, Nanobodies®, compounds, constructs and / or polypeptides of the invention and a carrier suitable for pulmonary delivery. . Accordingly, the present invention also relates to pharmaceutical compositions comprising the amino acid sequences, Nanobodies®, compounds, constructs and / or polypeptides of the invention and a carrier suitable for pulmonary delivery. Suitable carriers for pulmonary delivery are known in the art.

  The amino acid sequences, Nanobodies®, compounds, constructs and / or polypeptides of the invention may also be administered as pure drug microparticles or nanoparticles having a particle size and particle distribution that are advantageous for pulmonary delivery. Good.

  Accordingly, the present invention provides a pharmaceutical device suitable for pulmonary delivery of the amino acid sequences, Nanobodies®, compounds, constructs and / or polypeptides of the present invention and suitable for use of compositions comprising the same. Also concerns. The device can be an inhaler for liquids (eg, suspensions or droplets of solid microparticles) containing the amino acid sequences, Nanobodies®, compounds, constructs, and / or polypeptides of the invention. Preferably, the device is an aerosol comprising the amino acid sequence, Nanobody®, compound, construct, and / or polypeptide of the invention. The device may be a dry powder inhaler comprising the amino acid sequences, Nanobodies®, compounds, constructs and / or polypeptides of the invention in dry powder form.

  In a preferred method, administration to the lung tissue comprises inhaling the amino acid sequence, Nanobody®, compound, construct, and / or polypeptide of the invention and / or a composition comprising the same in an aerosol cloud. It is done by doing. According to the present invention, inhalation of the aerosol cloud can be performed by an inhalation device. The device comprises a formulation comprising an amino acid sequence, Nanobody (R), compound, construct, and / or polypeptide (and / or composition comprising) of the invention at an appropriate time during the mammalian inhalation cycle. Will generate an aerosol cloud of the desired particle size (particle distribution) containing the correct dose of the amino acid sequences, Nanobodies®, compounds, constructs, and / or polypeptides of the invention ("Pulmonary"). drug delivery ", Bechtold-Peters and Luessen, eds., ISBN 978-3-87193-322-6 page 125).

  In the context of the present invention, "aerosol" refers to a suspension of gaseous solid particulates or droplets (or a combination thereof), wherein for the purposes of the present invention, particles and / or droplets Includes amino acid sequences, Nanobodies®, compounds, constructs, and / or polypeptides of the invention.

The device can be used at any time during the inhalation cycle of a mammal to form, from a formulation, a desired particle containing a correct dose of an amino acid sequence, Nanobody®, compound, construct, and / or polypeptide of the invention. An aerosol cloud having a diameter (particle distribution) shall be generated. The following four requirements (formulation, particle size, time and dose) should be considered ("Pulmonary Drug Delivery", Bechtold-Peters and Luessen, eds., Ibid., Pages 125 and 126):
The formulation used for the device can be from aqueous solutions or suspensions used in nebulizers, to propellant-based solutions or suspensions used in metered dose inhalers, or even to dry powder inhalers. It can vary with specially designed powder mixtures. All of these various formulations require different principles for aerosol generation, which emphasizes the interdependence of devices and formulations.
Since the site of deposition of aerosol particles varies depending on their (aerodynamic) size and velocity, the desired particle size of the aerosol cloud will vary depending on the desired site of deposition in the lungs, Related to the purpose of treatment.
Since the aerosol cloud can be adjusted to be released at different times during the inhalation cycle occurring in mammals, the agents of the present invention (which deposit on the periphery of the lungs) require aerosol cloud at the beginning of the inhalation cycle. Is preferably released.
Dosages may vary widely, for example from a few micrograms to hundreds of micrograms or even several milligrams in a human, for example up to about 10 to 100 milligrams.

  Various inhalation systems are described, for example, in the reviews ("Pulmonary Drug Delivery", Bechtold-Peters and Luessen, eds., Supra) at pages 129-148, including nebulizers, metered dose inhalers, metered liquids. Including but not limited to inhalers and dry powder inhalers. Devices that take into account individual breathing patterns optimized for controlling inhalation manoeuvres may also be used ("Pulmonary Drug Delivery", AKITA on page 157 of Bechtold-Peters and Luessen, eds., Ibid.). (Registered trademark) technology).

However, in addition to the importance of this device for pulmonary delivery of the amino acid sequences, Nanobodies®, compounds, constructs, and / or polypeptides of the present invention, appropriate formulations achieve effective delivery Indispensable for. In principle, this can be achieved by using one of the following approaches:
Administration to the nasal cavity of an aqueous solution or suspension (for example, nasal drops) comprising the amino acid sequence, Nanobody®, compound, construct and / or polypeptide of the invention;
Nebulization of an aqueous solution or suspension containing the amino acid sequences, Nanobodies®, compounds, constructs, and / or polypeptides of the invention;
Atomization by liquefied high pressure gas; and dispersion of dry powder.

  Accordingly, there is a need to adapt and tailor the formulation of the amino acid sequences, Nanobodies®, compounds, constructs, and / or polypeptides of the invention to the chosen inhalation device. In addition to the amino acid sequences, Nanobodies®, compounds, constructs and / or polypeptides of the invention, suitable formulations, ie, excipients, are, for example, “Pulmonary Drug Delivery”, Bechtold-Peters and Luessen, eds , (Id.) In Chapter IV. In this regard, reference is also made to US Provisional Patent Application Ser. No. 61 / 303,447, filed Feb. 12, 2010 and filed by Ablynx N.V., entitled "Methods and compositions for the preparation of aerosols".

  The amount of the amino acid sequences, Nanobodies®, compounds, constructs and polypeptides of the invention required for use in therapy will depend only on the particular amino acid sequence, Nanobody®, compound, construct or polypeptide selected. Rather, it will also vary with the route of administration, the nature of the condition being treated, and the age and condition of the patient, and will ultimately be left to the discretion of the attending physician or clinician. Also, the dosage of the amino acid sequences, Nanobodies®, compounds, constructs and polypeptides of the invention will vary with the target host cell, tumor, tissue, graft or organ.

  The desired dose may conveniently be presented in a single dose or in divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses daily. The sub-dose itself may be further divided, eg, into a number of discrete, loosely spaced administrations, such as multiple inhalations from an insufflator or eye drops of multiple drops.

  Dosage regimes may include long-term daily treatment. By "long term" is meant a period of at least two weeks, preferably weeks, months, or years. The necessary changes in this dosage range can be determined by one of ordinary skill in the art using only routine experimentation taught herein. See Remington's Pharmaceutical Sciences (Martin, E.W., ed. 4), Mack Publishing Co., Easton, PA. The dosage can also be adjusted by the individual physician if any complications occur.

  The invention further relates to the application and use of the amino acid sequences, Nanobodies®, polypeptides, compounds, nucleic acids, host cells, products and compositions described herein, and to respiratory tract infections caused by hRSV. It relates to a method for prevention and / or treatment. Some preferred but non-limiting applications and uses will become apparent from the further description herein.

  The amino acid sequences, Nanobodies®, polypeptides, compounds and compositions of the present invention are generally used to block the interaction of hRSV protein F with target host cells and / or their membranes. to neutralize hRSV (various hRSV strains and / or escape mutants), to modulate, inhibit and / or prevent hRSV infectivity (of various hRSV strains and / or escape mutants); To regulate, inhibit and / or prevent fusion of (with various hRSV strains and / or escape mutants) to (the cell membrane of) target host cells, and / or (with various hRSV strains and / or escapes) It can be used to modulate, inhibit and / or prevent hRSV entry in target host cells (of the mutant).

  In one aspect, the amino acid sequences, Nanobodies®, polypeptides, compounds and compositions of the present invention can be prepared by any suitable method known per se, for example, using one of the assays described herein. Of protein F of hRSV with target host cells and / or their membranes under the same conditions but without the amino acid sequence, Nanobody® or polypeptide of the invention, as determined by a simple method As compared to at least 1%, preferably at least 5%, such as at least 10% or at least 25%, such as at least 50%, at least It can block 60%, at least 70%, at least 80%, or 90% or more.

  In another aspect, the amino acid sequences, Nanobodies®, polypeptides, compounds and compositions of the present invention can be any known per se, for example, using one of the assays described herein. HRSV infectivity is at least 1%, preferably at least 1%, as compared to neutralization of hRSV under the same conditions but without the amino acid sequence, Nanobody® or polypeptide of the invention, as determined by a suitable method. Neutralizes at least 5%, such as at least 10% or at least 25%, such as at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more.

  In the context of the present invention, "modulating" or "to modulate" generally refers to a virus, as measured using a suitable in vitro, cellular or in vivo assay. It means either reducing, preventing or inhibiting infectivity, fusion and / or viral entry, and / or reducing, preventing or inhibiting the biological pathway mediated by protein F of hRSV. In particular, "modulating" refers to an amino acid sequence of the invention, Nanobody (as determined using a suitable in vitro, cellular, or in vivo assay (such as those referred to herein)). ® or polypeptide in the same assay but under the same conditions but without the presence of normal (ie, native) viral infectivity, fusion and / or virus entry, and / or normal mediated by protein F of hRSV. At least 1%, preferably at least 5%, such as at least 10% or at least 25%, such as at least 50%, at least 60%, at least 70%, at least 80% compared to a natural (ie natural) biological pathway Or reduce virus infectivity, fusion and / or virus entry by 90% or more; It can mean either preventing or inhibiting and / or reducing, preventing or inhibiting a biological pathway mediated by protein F of hRSV.

  In one aspect, the amino acid sequences, Nanobodies®, polypeptides, compounds and compositions of the present invention can be prepared by any suitable method known per se, for example, using one of the assays described herein. At least 1%, preferably at least 5%, such as at least 5%, as compared to the infectivity under the same conditions but without the amino acid sequence, Nanobody® or polypeptide of the invention, as determined by an appropriate method. It can modulate, inhibit and / or prevent hRSV infectivity by 10% or at least 25%, such as at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more.

  As used herein, the term "virus entry" refers to any virus-mediated biological activity required to achieve virion binding and / or viral fusion with a target host cell. Include routes. Viral entry, which can be any virus-mediated biological pathway required to achieve virion binding to the target host cell and / or viral fusion with the target host cell, is herein described. Specificity of the amino acid sequences, Nanobodies®, polypeptides and / or compounds of the present invention as determined using a suitable in vitro, cellular or in vivo assay (such as those mentioned). It is encompassed by the present invention that it can be regulated and / or reduced and / or prevented and / or inhibited by binding. In particular, the amino acid sequences, Nanobodies®, polypeptides of the invention, as measured using a suitable in vitro, cellular or in vivo assay (such as those referred to herein). And / or the specific binding of the compound to hRSV protein F allows virus entry (which can be mediated by hRSV protein F) to occur in a normal (ie, native) virus (as defined herein). At least as compared to invasion, which can be mediated by protein F of hRSV in the same assay under the same conditions but without the amino acid sequence, Nanobody®, polypeptide and / or compound of the invention. 1%, preferably at least 5%, such as at least 10% or at least 25%, eg For example, at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more can be modulated, reduced, prevented or inhibited. Thus, the amino acid sequences, Nanobodies®, polypeptides and polypeptides of the invention, as measured using a suitable in vitro, cellular or in vivo assay (such as those mentioned herein). Also included is the ability to modulate and / or reduce and / or prevent and / or inhibit virus attachment and / or virus fusion by specific binding of the compound to hRSV protein F. In particular, the amino acid sequences, Nanobodies®, polypeptides of the invention, as measured using a suitable in vitro, cellular or in vivo assay (such as those referred to herein). And / or the specific binding of the compound to protein F of hRSV allows virus attachment and / or virus fusion (which can be mediated by protein F of hRSV) to occur with normal (ie, native) virus attachment and / or virus Fusion, which can be mediated by protein F of hRSV in the same assay under the same conditions but without the amino acid sequence, Nanobody®, polypeptide and / or compound of the invention 1%, preferably at least 5%, for example at least 10% or less It can modulate, reduce, prevent or inhibit at least 25%, such as at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more.

  In this regard, the amino acid sequences, Nanobodies®, polypeptides, compounds and compositions of the present invention can be prepared using, eg, one of the assays described herein. Trademark) or at least 1%, preferably at least 5%, such as at least 10% or at least 25%, such as at least 50%, as compared to entry into the targeted host cell under the same conditions but without the polypeptide. %, At least 60%, at least 70%, at least 80%, or 90% or more can modulate, inhibit and / or prevent hRSV entry into the targeted host cell.

  The amino acid sequences, Nanobodies®, polypeptides, compounds and compositions of the present invention are measured in any suitable manner known per se, for example using one of the assays described herein. At least 1% compared to the fusion of hRSV with (the cell membrane of) the target host cell under the same conditions but without the amino acid sequence, Nanobody® or polypeptide of the invention. Preferably at least 5%, such as at least 10% or at least 25%, such as at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more of the fusion of hRSV with (the cell membrane of) the target host cell. Can be regulated, inhibited and / or prevented.

  The multivalent (eg, divalent or trivalent) polypeptides of the present invention may comprise different genotypes, subtypes, viral escape mutants and / or hRSV compared to a monovalent amino acid sequence or Nanobody®. It showed improved affinity for the strain and / or improved cross-reactivity. In one aspect, a multivalent (eg, divalent or trivalent) polypeptide of the present invention can bind to various RSV strains (eg, Long, A-2 and / or B-1). In yet another aspect, the multivalent (eg, divalent or trivalent) polypeptides of the present invention may comprise a variety of hRSV variants (eg, as described in Lopez et al. 1998, J. Virol. 72: 6922-6928). It can bind to an escape mutant and / or an escape mutant specific for antigenic site II, antigenic sites IV-VI, or a combination of both antigenic sites.

  Accordingly, the present invention also relates to the use of a multivalent (eg trivalent, divalent) polypeptide of the invention and / or a pharmaceutical composition comprising the same for the binding and / or neutralization of various hRSV strains. . In a preferred embodiment, the bivalent humanized and / or sequence-optimized NC41 Nanobody® (eg, selected from SEQ ID NO: 60-SEQ ID NO: 76, SEQ ID NO: 138-SEQ ID NO: 141, and SEQ ID NO: 146-SEQ ID NO: 157) Bivalent polypeptides containing two Nanobodies®). In another preferred embodiment, a trivalent humanized and / or sequence-optimized NC41 Nanobody® (e.g., from SEQ ID NO: 60-SEQ ID NO: 76, SEQ ID NO: 138-SEQ ID NO: 141, and SEQ ID NO: 146-SEQ ID NO: 157) A trivalent polypeptide comprising three of the selected Nanobodies® is used. In another preferred embodiment, one of SEQ ID NOs: 77-79, SEQ ID NOs: 142-145, and SEQ ID NOs: 158-165 is used.

  The present invention relates to polyvalent (eg trivalent, bivalent) polypeptides of the invention for binding and / or neutralization of one or more escape mutants of hRSV, and / or pharmaceutical compositions comprising the same. Also relates to the use of. In a preferred embodiment, a bivalent humanized NC41 Nanobody® (eg, a Nanobody® selected from SEQ ID NO: 60-SEQ ID NO: 76, SEQ ID NO: 138-SEQ ID NO: 141, and SEQ ID NO: 146-SEQ ID NO: 157) And two divalent polypeptides). In another preferred embodiment, a trivalent humanized NC41 Nanobody® (eg, a Nanobody selected from SEQ ID NO: 60-SEQ ID NO: 76, SEQ ID NO: 138-SEQ ID NO: 141, and SEQ ID NO: 146-SEQ ID NO: 157 (registered Trademark)) is used. In another preferred embodiment, one of SEQ ID NOs: 77-79, SEQ ID NOs: 142-145, and SEQ ID NOs: 158-165 is used.

  The present invention provides a method for preventing and / or treating at least one viral disease, comprising a pharmaceutically active amount of an amino acid sequence of the present invention, a Nanobody® of the present invention, a polypeptide of the present invention, It also relates to a method comprising administering a compound or construct of the invention and / or a pharmaceutical composition comprising them to a subject in need thereof.

  Accordingly, the amino acid sequences, Nanobodies®, polypeptides, compounds and compositions of the present invention can be used for the prevention and / or treatment of diseases and disorders associated with hRSV infection. Examples of such diseases and disorders associated with hRSV infection will be apparent to those of skill in the art based on the disclosure herein, including, for example, the following diseases and disorders: respiratory diseases, upper respiratory tract infections Lower respiratory tract infections, bronchiolitis (inflammation of the peripheral airways in the lungs), pneumonia, dyspnea, cough, (recurrent) wheezing and / or asthma.

  Accordingly, the present invention relates to respiratory diseases, upper respiratory tract infections, lower respiratory tract infections, bronchiolitis (inflammation of the peripheral airways in the lungs), pneumonia, dyspnea, cough, (recurrent) wheeze and / or Or a method for preventing and / or treating asthma, comprising a pharmaceutically active amount of at least one amino acid sequence of the invention, a Nanobody® of the invention, a polypeptide of the invention, a compound of the invention Or a construct, or a monovalent construct of the invention, or a composition of the invention, to a subject in need thereof.

  The present invention relates to the prevention and / or prevention of respiratory diseases, upper respiratory tract infections, lower respiratory tract infections, bronchiolitis (inflammation of the peripheral airways in the lungs), pneumonia, dyspnea, cough, (recurrent) wheezing and / or asthma. Or the amino acid sequence of the invention, the Nanobody® of the invention, the invention in the preparation of a pharmaceutical composition for therapy and / or for use in one or more of the methods described herein. Or a compound or construct of the invention, or a monovalent construct of the invention.

  The present invention relates to the prevention and / or prevention of respiratory diseases, upper respiratory tract infections, lower respiratory tract infections, bronchiolitis (inflammation of the peripheral airways in the lungs), pneumonia, dyspnea, cough, (recurrent) wheezing and / or asthma. Or an amino acid sequence of the invention, a Nanobody® of the invention, a polypeptide of the invention, a compound or construct of the invention, or a monovalent construct of the invention for therapy.

  In the context of the present invention, the term “prevention and / or treatment” includes not only preventing and / or treating a disease, but also generally preventing the onset of the disease, delaying or regressing the progression of the disease. Causing, preventing or delaying the onset of one or more symptoms associated with the disease, reducing and / or alleviating one or more symptoms associated with the disease, any disease or / and any related thereto. Reducing the severity and / or duration of the symptoms, and / or preventing a further increase in the severity of the disease and / or any symptoms associated therewith, preventing, reducing or reducing any physiological damage caused by the disease. It also includes regression and generally any pharmacological effects that are beneficial to the patient being treated.

  The subject to be treated can be any warm-blooded animal, but is specifically a mammal, and more specifically a human. As will be apparent to those of skill in the art, the subject to be treated can be a human who is particularly suffering from or at risk for the diseases and disorders mentioned herein.

  More specifically, the present invention is a method for preventing and / or treating an infection caused by hRSV, comprising a pharmaceutically active amount of a polyvalent (eg trivalent or divalent) polypeptide or compound of the invention. And / or administering a pharmaceutical composition comprising the same to a subject in need thereof. More specifically, the present invention is a method for preventing and / or treating an infection caused by hRSV, which requires a pharmaceutically active amount of a divalent compound or polypeptide of the present invention. The method may comprise administering to the subject. More specifically, the present invention is a method for preventing and / or treating an infection caused by hRSV, comprising a pharmaceutically active amount of a bivalent humanized and / or sequence-optimized NC41 Nanobody®. (For example, a bivalent polypeptide containing two Nanobodies (registered trademark) selected from SEQ ID NO: 60 to SEQ ID NO: 76, SEQ ID NO: 138 to SEQ ID NO: 141, and SEQ ID NO: 146 to SEQ ID NO: 157). The method may comprise administering to a subject in need thereof. More specifically, the present invention is a method for preventing and / or treating an infection caused by hRSV, which requires a pharmaceutically active amount of a trivalent compound or polypeptide of the present invention. The method may comprise administering to the subject. More specifically, the present invention is a method of preventing and / or treating an infection due to hRSV, comprising a pharmaceutically active amount of a trivalent humanized NC41 Nanobody® (eg, SEQ ID NO: 60). To SEQ ID NO: 76, SEQ ID NO: 138 to SEQ ID NO: 141, and SEQ ID NO: 146 to SEQ ID NO: 157) to a subject in need thereof. Administration can be provided. More specifically, the present invention is a method for preventing and / or treating an infection caused by hRSV, comprising a pharmaceutically active amount of SEQ ID NO: 77 to SEQ ID NO: 79, SEQ ID NO: 142 to SEQ ID NO: 145 and SEQ ID NO: No. 158 to SEQ ID NO: 165 to a subject in need thereof.

  More specifically, the present invention relates to a method for preventing and / or treating an infection caused by hRSV, comprising a pharmaceutically active amount of an amino acid sequence of the present invention, a Nanobody® of the present invention, The method may comprise administering a polypeptide of the invention and / or a pharmaceutical composition comprising the same to lung tissue of a subject in need thereof.

  In another aspect, the present invention relates to a method for immunotherapy, in particular passive immunotherapy, comprising a pharmaceutically active amount of an amino acid sequence of the invention, a Nanobody® of the invention, a polypeptide of the invention. Administering a compound or construct of the invention and / or a pharmaceutical composition comprising them to a subject suffering from or at risk for the diseases and disorders mentioned herein. On the method.

  In the above methods, the amino acid sequences, Nanobodies®, compounds or constructs, and / or polypeptides of the invention, and / or compositions containing them, may comprise a particular pharmaceutical formulation or composition used. It can be administered in any suitable manner depending on the substance. Thus, the amino acid sequences, Nanobodies® and / or polypeptides of the invention and / or compositions comprising them can be used, for example, in certain pharmaceutical formulations or compositions also used here. Orally, intraperitoneally (eg, intravenously, subcutaneously, intramuscularly, or via any other route of administration bypassing the gastrointestinal tract), intranasally, transdermally, topically, using suppositories, as appropriate Can be administered by inhalation. The clinician will determine the preferred route of administration, and the suitable pharmaceutical formulation or composition to be used for such administration, depending on the disease or disorder to be prevented or treated, and other factors known to the clinician. Things could be selected.

Thus, in general, the amino acid sequences, Nanobodies®, compounds or constructs, and polypeptides, and / or compositions comprising them, according to the present invention can be administered in any suitable manner, including, but not limited to, The amino acid sequences, Nanobodies®, compounds or constructs and polypeptides according to the invention, and compositions comprising them, can be administered intranasally and / or by inhalation and / or any other suitable for pulmonary delivery It can be administered in any form. Methods for pulmonary and / or nasal and / or inhalation delivery of Nanobodies®, amino acid sequences, compounds or constructs, and / or polypeptides of the invention are known to those of skill in the art and include, for example, Binghe. Handbook "Drug Delivery: Principles and Applications" (2005) (Eds. Wiley Interscience (John Wiley & Sons)) by Wang, Teruna Siahaan and Richard Soltero in Ablynx N.V., entitled "Intranasaldelivery of polypeptides and proteins" in International Publication No. 08/049897 of the international application, Rosenfeld GC, due to the Loose-Mitchell DS in ^{"Pharmacology PreTest TM Self-Assessment and} Review" (11 th Edition), and Lippincott Williams & Wilkins, New York, Shlafer M. McGraw -Hill Medical Publishing Division, New York, Yang KY, Graff LR, Caughey AB Blueprints Pharmacology, are described in accordance "Pharmacology" (3 ^rd Edition) to BlackwellPublishing.

  Accordingly, the present invention provides a method of administering an effective amount of an amino acid sequence, Nanobody®, compound or construct, and / or polypeptide of the invention, and / or a composition comprising the same, comprising: A method comprising administering to a lung tissue a sequence, Nanobody®, compound or construct, and / or polypeptide, and / or a composition comprising the same. In such a method, the amino acid sequence, Nanobody®, compound or construct, and / or polypeptide, and / or composition comprising the same, can be prepared by any method known in the art for pulmonary delivery, for example, Administration can be by use of an inhaler or intranasal delivery device or an aerosol.

  In a preferred embodiment of the invention, the amino acid sequence, Nanobody (R), compound or construct, and / or polypeptide binds to and / or neutralizes a virus present in lung tissue. Preferably, in such a method for pulmonary delivery, at least 5%, preferably at least 10%, 20%, 30%, 40%, more preferably at least 50%, 60%, 70%, even more preferably at least 80% % Or more of the amino acid sequences, Nanobodies®, compounds or constructs, and / or polypeptides of the invention are stable in lung tissue for at least 24 hours, preferably at least 48 hours, more preferably at least 72 hours. .

  Surprisingly, it has been found that the amino acid sequences, Nanobodies®, compounds or constructs, and / or polypeptides of the invention have long-term stability in lung tissue. For example, it has been found that Nanobodies® that are hRSV directional remain functional in the lung for at least 48 hours ("Amino acid sequences directed against envelope proteins of a virus and polypeptides comprising the same for the treatment"). (See International Application No. PCT / EP2009 / 056975, filed June 5, 2009, filed by Ablynx NV, entitled "Viral diseases." Therefore, treatment intervals are once a day, once every two days, once every three days, once every four days, once every five days, once every six days, or once a week. Embodiments of the invention will be able to take the putative long-term stability of the amino acid sequences, Nanobodies®, compounds or constructs, and / or polypeptides of the invention.

  Accordingly, the present invention provides a method of delivering an amino acid sequence, Nanobody®, compound or construct, and / or polypeptide of the invention to lung tissue of a subject without inactivation, comprising: A pulmonary administration of the amino acid sequence, Nanobody®, compound or construct, and / or polypeptide of the invention to said subject.

  The present invention relates to a method for preventing and / or treating hRSV infection, comprising a pharmaceutically active amount of an amino acid sequence of the present invention, a Nanobody® of the present invention, a polypeptide of the present invention, And / or a pharmaceutical composition comprising the same or a pharmaceutical composition comprising the same to a lung tissue of a subject in need thereof.

  More specifically, the invention relates to respiratory diseases, upper respiratory tract infections, lower respiratory tract infections, bronchiolitis (inflammation of the peripheral airways in the lungs), pneumonia, dyspnea, cough, (recurrent) wheezing and / or A method of preventing and / or treating asthma, comprising a pharmaceutically active amount of an amino acid sequence of the invention, a Nanobody® of the invention, a polypeptide of the invention, or a compound or construct of the invention. And / or administering a pharmaceutical composition comprising them to the lung tissue of a subject in need thereof.

  More specifically, the present invention is a method of preventing and / or treating an infection caused by hRSV, comprising a pharmaceutically active amount of a polyvalent (eg trivalent, divalent) polypeptide or compound of the invention. And / or administering a pharmaceutical composition comprising them to the lung tissue of a subject in need thereof. More specifically, the present invention is a method for preventing and / or treating an infection caused by hRSV, which requires a pharmaceutically active amount of a divalent compound or polypeptide of the present invention. The method may comprise administering to the lung tissue of the subject. More specifically, the present invention is a method of preventing and / or treating an infection caused by hRSV, comprising a pharmaceutically active amount of a bivalent humanized NC41 Nanobody® (eg, SEQ ID NO: 60). ~ A divalent polypeptide comprising two Nanobodies (registered trademark) selected from SEQ ID NO: 76, SEQ ID NO: 138 to SEQ ID NO: 141, and SEQ ID NO: 146 to SEQ ID NO: 157) of a subject in need thereof The method may comprise administering to lung tissue. More specifically, the present invention is a method for preventing and / or treating an infection caused by hRSV, which requires a pharmaceutically active amount of a trivalent compound or polypeptide of the present invention. The method may comprise administering to the lung tissue of the subject. More specifically, the present invention is a method of preventing and / or treating an infection due to hRSV, comprising a pharmaceutically active amount of a trivalent humanized NC41 Nanobody® (eg, SEQ ID NO: 60). To SEQ ID NO: 76, SEQ ID NO: 138 to SEQ ID NO: 141, and SEQ ID NO: 146 to SEQ ID NO: 157) to a subject in need thereof. The method may comprise administering to lung tissue. More specifically, the present invention is a method for preventing and / or treating an infection caused by hRSV, comprising a pharmaceutically active amount of SEQ ID NO: 77 to SEQ ID NO: 79, SEQ ID NO: 142 to SEQ ID NO: 145 and SEQ ID NO: No. 158 to SEQ ID NO: 165 to a lung tissue of a subject in need thereof.

Also, for example, without limitation, amino acid sequences, Nanobodies®, compounds or constructs, and polypeptides according to the present invention, and compositions comprising them, can be administered intramuscularly and / or to any suitable brain. The delivery form can be administered in any suitable delivery form, eg, the amino acid sequences, Nanobodies®, polypeptides, compounds or constructs described above, and compositions comprising them, that are transported across the blood-brain barrier. . Such methods for intramuscular delivery of Nanobodies®, amino acid sequences, and / or polypeptides of the invention and / or any suitable form of delivery to the brain are known to those of skill in the art and are described, for example, in Binghe Wang, "Pharmacology PreTest ^TM Self-Assessment and Review" by Rosenfeld GC, Loose-Mitchell DS in "Drug Delivery: Principles and Applications" (2005) (Eds. Wiley Interscience (John Wiley & Sons)) by Teruna Siahaan and Richard Soltero to (11 ^th Edition), and Lippincott Williams & Wilkins, New York, Shlafer M. McGraw-Hill Medical Publishing Division, New York, Yang KY, Graff LR, Caughey AB Blueprints Pharmacology, according to ^{Blackwell Publishing "Pharmacology" (3 rd} Edition )It is described in.

  The amino acid sequence, Nanobody (registered trademark), compound or construct, and / or polypeptide of the present invention, and / or a composition containing the same is suitable for the prevention and / or treatment of a disease or disorder to be prevented or treated. Administered according to schedule. The clinician generally knows the disease or disorder to be prevented or treated, the severity of the disease to be treated and / or the severity of its symptoms, the particular amino acid sequence of the invention used, the Nanobody®, the compound Or the construct or polypeptide, the particular route of administration, and the pharmaceutical formulation or composition used, factors such as the patient's age, sex, weight, diet, general condition, and the like, as well as those known to the clinician. Depending on the factors, a suitable treatment regime will be able to be determined.

  Generally, a treatment regimen will include one or more pharmaceutically effective amounts or doses of one or more amino acid sequences, Nanobodies®, compounds or constructs, and / or polypeptides of the invention, or Would include administration of one or more compositions comprising The specific dosage (s) or dosages can still be determined by the clinician based on the above factors.

  In general, with respect to the prevention and / or treatment of the diseases and disorders referred to herein, as well as the particular disease or disorder to be treated, the particular amino acid sequence, Nanobody (R), compound or construct of the present invention used. Amino acid sequences, Nanobodies®, compounds or constructs, and polypeptides of the invention, depending on the potency of the polypeptide, the particular route of administration, and the particular pharmaceutical formulation or composition used. Generally is from 1 gram / kg (body weight) / day to 1 milligram / kg (body weight) / day, preferably from 0.1 gram / kg (body weight) / day to 10 mg / kg (body weight) / day, most preferably Is 0.01 gram / kg (body weight) / day to 100 mg / kg (body weight) / day, for example, about 0.1 mg / kg (body weight) / day, 0.5 mg / kg (body weight) / Day, 1 mg / kg (body weight) / day, 2 mg / kg (body weight) / day, 5 mg / kg (body weight) / day, or 10 mg / kg (body weight) / day, continuously It will be administered (eg, by infusion), as a single daily dose, or as multiple divided doses throughout the day. Amino acid sequences, Nanobodies®, compounds or constructs, and polypeptides of the invention containing moieties that increase half-life may be administered once or twice a month, from 1 milligram / kg (body weight) to 100 milligrams / kg. (Body weight), preferably 1 mg / kg (body weight) to 50 mg / kg (body weight), for example about 10 mg / kg (body weight), 15 mg / kg (body weight), 20 mg / kg (body weight), or 30 mg / kg (body weight). It can be administered in amounts of milligrams / kg (body weight). A clinician will generally be able to determine a suitable daily dose depending on the factors mentioned herein. It will be clear that in certain cases, the clinician may choose to deviate from these amounts, for example based on the above factors and the clinician's professional judgment. In general, comparisons to similar targets administered via essentially similar routes, taking into account differences in affinity / binding activity, efficacy, biodistribution, half-life and similar factors known to those skilled in the art. Some guidance on dosage can be obtained from the amounts normally administered for possible conventional antibodies or antibody fragments.

  When administering amino acid sequences, Nanobodies®, compounds or constructs, and / or polypeptides, and / or compositions comprising them to lung tissue, the treatment regimen may be once or twice daily, preferably 1 hour. It can be once daily, or once every 2, 3, 4, 5, 6, or 7 days.

  Typically, the above methods will use a single amino acid sequence, Nanobody®, compound or construct, or polypeptide of the invention. However, it is also within the scope of the present invention to use a combination of two or more amino acid sequences, Nanobodies®, compounds or constructs, and / or polypeptides of the present invention.

  The Nanobodies®, amino acid sequences, compounds or constructs, and polypeptides of the invention can be combined with one or more additional pharmaceutically active compounds or components, ie, to produce or produce a synergistic effect. It can also be used as a complex treatment plan that may not be present. In this case, too, the clinician will be able to select such further compounds or ingredients, as well as a suitable combined treatment regimen, based on the above factors and the clinician's professional judgment.

  In particular, the amino acid sequences, Nanobodies®, compounds or constructs, and polypeptides of the invention may be used or used in the prevention and / or treatment of the diseases and disorders described herein. It can be used in combination with a pharmaceutically active compound or ingredient, which may or may not result in a synergistic effect. Examples of such compounds and components, as well as routes, methods and pharmaceutical formulations or compositions for administering them, will be apparent to the clinician.

  When two or more substances or components are used as part of a combined treatment regimen, they may be administered at the same time or at different times, via similar routes of administration or through different routes of administration (eg, At the same time, sequentially or according to an alternating schedule). If the substances or components are to be administered simultaneously via the same route of administration, as will be apparent to those skilled in the art, different pharmaceutical formulations or compositions or combined pharmaceutical formulations or compositions May be administered as part of

  Also, if more than one active substance or ingredient is to be used as part of a combined treatment regimen, the substance may be used in amounts similar to those used when the compound or ingredient is used alone, and in accordance with the same regimen. Or each of the components may be administered, and such combinations may or may not produce a synergistic effect. However, if a combination of two or more active substances or ingredients produces a synergistic effect, the amount of one, more or all of the administered substance or ingredient may be reduced while achieving the desired therapeutic effect. It may also be possible to reduce it. This may, for example, still achieve the desired pharmacological or therapeutic effect and avoid, limit or avoid any undesirable side effects associated with the use of one or more substances or ingredients when used in common amounts. It may be useful to reduce.

  The effectiveness of the treatment regimen used in accordance with the present invention can be determined and / or pursued in any manner known per se for the disease or disorder involved, as will be apparent to the clinician. The clinician also changes or modifies the particular treatment regimen as needed and on a case-by-case basis, thereby achieving the desired therapeutic effect, avoiding, limiting or reducing undesired side effects, and / or A suitable balance between achieving the desired therapeutic effect on the one hand and avoiding, limiting or reducing undesired side effects on the other hand can be achieved.

  Generally, the treatment plan is continued until the desired therapeutic effect is achieved and / or as long as the desired therapeutic effect needs to be maintained. Again, this can be determined by the clinician.

  In another aspect, the invention relates to a pharmaceutical composition for preventing and / or treating at least one viral disease; and / or for use in one or more of the treatment methods mentioned herein. It relates to the use of the amino acid sequences, Nanobodies®, compounds or constructs or polypeptides of the invention in the preparation.

  The subject to be treated can be any warm-blooded animal, but is specifically a mammal, and more specifically a human. As will be apparent to those of skill in the art, the subject to be treated is a human who is particularly afflicted with or at risk for the diseases and disorders mentioned herein.

  The present invention also provides for preventing and / or preventing at least one disease or disorder that can be prevented and / or treated by administering an amino acid sequence, Nanobody®, compound or construct, or polypeptide of the invention to a patient. Or the use of an amino acid sequence, Nanobody®, compound or construct or polypeptide of the invention in the preparation of a pharmaceutical composition for treatment.

  More specifically, the present invention relates to pharmaceutical compositions for preventing and / or treating viral diseases, and in particular for preventing and treating one or more diseases and disorders listed herein. The use of an amino acid sequence, Nanobody®, compound or construct or polypeptide of the invention in the preparation of

  Also in such pharmaceutical compositions, one or more of the amino acid sequences, Nanobodies®, compounds or constructs, or polypeptides of the present invention may be linked to one or more, such as those referred to herein. May be suitably combined with other active ingredients.

  Further uses of the amino acid sequences, Nanobodies®, polypeptides, nucleic acids, genetic constructs, and hosts and host cells of the present invention will be apparent to those skilled in the art based on the disclosure herein. For example, without limitation, an amino acid sequence of the invention can be linked to a suitable carrier or solid support, thereby purifying the viral envelope protein from the composition and preparations containing the composition. A medium is provided which can be used in a manner known per se. Derivatives of the amino acid sequences of the present invention that include a suitable detectable label may also be used as markers to determine (qualitatively or quantitatively) the presence of the viral envelope protein in the composition or preparation, or (In combination with a suitable cell sorting technique) can be used as a marker to selectively detect the presence of the viral envelope protein on the surface of a cell or tissue.

  The invention will now be further described by the following non-limiting preferred embodiments, examples and figures.

  The entire contents of all references (including references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are expressly incorporated by reference, especially with respect to the teachings referred to above.

Aspect
Aspect A-1. has directivity and / or specifically binds to protein F of hRSV,
a) SEQ ID NO: 102,
b) a stretch of amino acid residues having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 102,
i) the amino acid residue stretch has aspartic acid (Asp, D) at position 6 (position 54 determined by Kabat numbering),
ii) the amino acid sequence comprising the amino acid residue stretch has the same, substantially the same or higher affinity as compared to the amino acid sequence comprising the amino acid residue stretch having no three, two or one amino acid difference ( The affinity is as measured by surface plasmon resonance) and binds to protein F of hRSV and / or the amino acid sequence comprising said stretch of amino acid residues differs by three, two or one amino acid An amino acid selected from an amino acid residue stretch that has the same, approximately the same, or a higher potency (as defined herein) as compared to an amino acid sequence comprising the above amino acid residue stretch without An amino acid sequence comprising at least one residue stretch.
Aspect A-2. Including two or more amino acid residue stretches, one stretch is
a) SEQ ID NO: 102,
b) a stretch of amino acid residues having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 102,
i) the amino acid residue stretch has aspartic acid (Asp, D) at position 6 (position 54 determined by Kabat numbering),
ii) the amino acid sequence comprising the amino acid residue stretch has the same, substantially the same or higher affinity as compared to the amino acid sequence comprising the amino acid residue stretch having no three, two or one amino acid difference ( The affinity is as measured by surface plasmon resonance) and binds to protein F of hRSV and / or the amino acid sequence comprising said stretch of amino acid residues differs by three, two or one amino acid Is selected from amino acid residue stretches that have the same, approximately the same, or higher potency (as defined herein) as compared to the amino acid sequence comprising the amino acid residue stretch without
At least one stretch,
c) SEQ ID NO: 98;
d) An amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 98, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (as said affinity is measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference the amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Stretches of amino acid residues, which shall have about the same or higher potency (as defined herein), e) Column number 121, and
f) an amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 121, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (such as measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference an amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Select from stretches of amino acid residues that should have about the same or higher potency (as defined herein). Wherein the amino acid residue stretch corresponding to one of a) and b) is such that it is always present in the amino acid sequence of the invention, and the second amino acid residue stretch is c), d), e A) the amino acid sequence according to aspect A-1, which is as selected from one of)) and f).
Aspect A-3. The first amino acid residue stretch comprises three or more amino acid residue stretches,
a) SEQ ID NO: 98,
b) An amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 98, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (such as measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference an amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Consisting of stretches of amino acid residues, which should have about the same or higher potency (as defined herein) It is selected from,
The second amino acid residue stretch is
c) SEQ ID NO: 102,
d) a stretch of amino acid residues having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 102,
i) the amino acid residue stretch has aspartic acid (Asp, D) at position 6 (position 54 determined by Kabat numbering),
ii) the amino acid sequence comprising the amino acid residue stretch has the same, substantially the same or higher affinity as compared to the amino acid sequence comprising the amino acid residue stretch having no three, two or one amino acid difference ( The affinity is as measured by surface plasmon resonance) and binds to protein F of hRSV and / or the amino acid sequence comprising the amino acid residue stretch has three, two or one amino acid differences. Selected from the group consisting of amino acid residue stretches that have the same, approximately the same, or higher potency (as defined herein) as compared to an amino acid sequence comprising the above amino acid residue stretches without And
The third amino acid residue stretch is
e) SEQ ID NO: 121, and
f) an amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 121, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (such as measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference an amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Stretches of amino acid residues which should be of about the same or higher potency (as defined herein). It is selected from the group, the amino acid sequence according to aspect A-1 or A-2.
Aspect A-4. Any of embodiments A-1 to A-3, which specifically binds to antigenic site II on protein F of hRSV and / or competes with SYNAGIS® for binding of hRSV to protein F The amino acid sequence described.
Aspect A-5. The amino acid sequence according to any one of aspects A-1 to A-4, which is a natural amino acid sequence (from any suitable species) or a synthetic or semi-synthetic amino acid sequence.
Aspect A-6. The amino acid sequence according to any one of aspects A-1 to A-5, which comprises an immunoglobulin fold or is capable of forming an immunoglobulin fold under suitable conditions.
Aspect A-7. The amino acid sequence according to any one of aspects A-1 to A-6, which is an immunoglobulin sequence.
Aspect A-8. The amino acid sequence according to any one of aspects A-1 to A-7, which is a natural amino acid sequence (from any suitable species) or a synthetic or semi-synthetic amino acid sequence.
Aspect A-9. The amino acid sequence according to any one of aspects A-1 to A-8, which is a humanized immunoglobulin sequence, a camelized immunoglobulin sequence, or an immunoglobulin sequence obtained by a technique such as affinity maturation.
Aspect A-10. Heavy chain variable domain sequence (eg, V _H The amino acid sequence according to any one of aspects A-1 to A-9, consisting essentially of the sequence
Aspect A-11. Aspects A-1 to A-10, consisting essentially of a heavy chain variable domain sequence derived from a conventional four chain antibody, or consisting essentially of a heavy chain variable domain sequence derived from a heavy chain antibody. Amino acid sequence of
Aspect A-12. Domain antibodies (or amino acid sequences suitable for use as domain antibodies), single domain antibodies (or amino acid sequences suitable for use as single domain antibodies), "dAbs" (or amino acid sequences suitable for use as dAbs) ) Or Nanobody (registered trademark) (V _HH An amino acid sequence according to any one of aspects A-1 to A-11, comprising, but not limited to, a sequence.
Embodiment A-1 3. Consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR2 comprises
a) SEQ ID NO: 102,
b) a stretch of amino acid residues having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 102,
i) the amino acid residue stretch has aspartic acid (Asp, D) at position 6 (position 54 determined by Kabat numbering),
ii) the amino acid sequence comprising the amino acid residue stretch has the same, substantially the same or higher affinity as compared to the amino acid sequence comprising the amino acid residue stretch having no three, two or one amino acid difference ( The affinity is as measured by surface plasmon resonance) and binds to protein F of hRSV and / or the amino acid sequence comprising said stretch of amino acid residues differs by three, two or one amino acid Selected from amino acid residue stretches that have the same, approximately the same, or higher potency (as defined herein) as compared to an amino acid sequence comprising the amino acid residue stretch without The amino acid sequence according to any one of aspects A-1 to A-12.
Aspect A-14.4 consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR2 comprises
a) SEQ ID NO: 102, or
b) a stretch of amino acid residues having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 102,
i) the amino acid residue stretch has aspartic acid (Asp, D) at position 6 (position 54 determined by Kabat numbering),
ii) the amino acid sequence comprising the amino acid residue stretch has the same, substantially the same or higher affinity as compared to the amino acid sequence comprising the amino acid residue stretch having no three, two or one amino acid difference ( The affinity is as measured by surface plasmon resonance) and binds to protein F of hRSV and / or the amino acid sequence comprising said stretch of amino acid residues differs by three, two or one amino acid 31 from the group consisting of amino acid residue stretches, which shall have the same, approximately the same, or higher potency (as defined herein) as compared to the amino acid sequence comprising said amino acid residue stretches without Or at least one of the CDR3s
c) SEQ ID NO: 98;
d) An amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 98, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (as said affinity is measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference the amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Consisting of stretches of amino acid residues, which should have about the same or higher potency (as defined herein) Is selected from CDR1, and / or
e) SEQ ID NO: 121, and
f) an amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 121, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (such as measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference an amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Stretches of amino acid residues which should be of about the same or higher potency (as defined herein). CDR3 is selected from the group
The amino acid sequence according to aspect A-13, which is selected from the group consisting of:
Aspect A-15.4 consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR1 is
a) SEQ ID NO: 98,
b) An amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 98, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (such as measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference an amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Consisting of stretches of amino acid residues, which should have about the same or higher potency (as defined herein) It is selected from,
CDR2 is
c) SEQ ID NO: 102,
d) a stretch of amino acid residues having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 102,
i) the amino acid residue stretch has aspartic acid (Asp, D) at position 6 (position 54 determined by Kabat numbering),
ii) the amino acid sequence comprising the amino acid residue stretch has the same, substantially the same or higher affinity as compared to the amino acid sequence comprising the amino acid residue stretch having no three, two or one amino acid difference ( The affinity is as measured by surface plasmon resonance) and binds to protein F of hRSV and / or the amino acid sequence comprising the amino acid residue stretch has three, two or one amino acid differences. Selected from the group consisting of amino acid residue stretches that have the same, approximately the same, or higher potency (as defined herein) as compared to an amino acid sequence comprising the above amino acid residue stretches without And
CDR3 is
e) SEQ ID NO: 121,
f) an amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 121, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (such as measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference an amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Stretches of amino acid residues which should be of about the same or higher potency (as defined herein). It is selected from the group, the amino acid sequence according to aspect A-13 or A-14.
Aspect A-16. The amino acid sequence according to any one of aspects A-1 to A-15, comprising at least SEQ ID NO: 102.
Aspect A-17. At least SEQ ID NO: 102,
a) SEQ ID NO: 98,
b) an amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 98, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (as said affinity is measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference the amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; An amino acid residue stretch, which shall have about the same or higher potency (as defined herein);
c) SEQ ID NO: 121, and
d) An amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 121, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (as said affinity is measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference the amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Select from stretches of amino acid residues that should have about the same or higher potency (as defined herein). Is the, and at least one amino acid residue stretches (CDR sequence), the amino acid sequence according to aspect A-16.
Aspect A-18. At least SEQ ID NO: 102,
a) SEQ ID NO: 98, and
b) an amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 98, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (as said affinity is measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference the amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Select from stretches of amino acid residues that should have about the same or higher potency (as defined herein) CDR1 and sequence, c) SEQ ID NO: 121 and,
d) An amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 121, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (as said affinity is measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference the amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Select from stretches of amino acid residues that should have about the same or higher potency (as defined herein). Is the and a CDR3 sequence, the amino acid sequence according to aspect A-16 or A-17.
Aspect A-19. Amino acid according to any one of aspects A-16 to A-18, comprising at least SEQ ID NO: 102 and at least one stretch of amino acid residues (CDR sequence) selected from SEQ ID NO: 98 and SEQ ID NO: 121 Array.
Aspect A-20. The amino acid sequence according to any one of aspects A-16 to A-19, comprising SEQ ID NO: 98, SEQ ID NO: 102, and SEQ ID NO: 121.
Aspect A-21.
a) at least 80% amino acid identity (determining the degree of amino acid identity) to at least one of the amino acid sequences of SEQ ID NOs: 60 to 76, SEQ ID NOs: 138 to 141, and 146 to 157; To ignore the amino acid residues that form the CDR sequence).
b) Preferably one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to Kabat numbering are international Selected from the characteristic residues referred to in Tables A-3 to A-8 of Publication 08/020079.
The amino acid sequence according to any one of aspects A-1 to A-20, consisting essentially of Nanobody®.
Aspect A-22. The amino acid sequence according to any one of aspects A-1 to A-21, consisting essentially of Humanized Nanobody®.
Aspect A-23. can bind to protein F of hRSV (as defined herein);
i) a humanized variant of the amino acid sequence of SEQ ID NO: 5 (see Table A-1), and / or
ii) the amino acid sequence of SEQ ID NO: 5 (see Table A-1) and / or SEQ ID NO: 60-SEQ ID NO: 76, SEQ ID NO: 138-SEQ ID NO: 141, and SEQ ID NO: 146-SEQ ID NO: 157 (Table A-4) ) Has at least 80% amino acid identity (ignoring the amino acid residues forming the CDR sequence to determine the degree of amino acid identity) with at least one of the amino acid sequences of
iii) Preferably one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to Kabat numbering are international Selected from the characteristic residues referred to in Tables A-3 to A-8 of Publication 08/020079.
The amino acid sequence of aspect A-22, consisting essentially of Humanized Nanobody®.
Aspect A-24. The amino acid sequence according to any one of aspects A-1 to A-23, which is in essentially isolated form.
Aspect A-25. The amino acid sequence according to any one of aspects A-1 to A-24, for administration to a subject, wherein the amino acid sequence is not naturally occurring in the subject.
Aspect A-26. Dissociation constant (K of 1000 nM to 1 nM or less, preferably 100 nM to 1 nM or less, more preferably 10 nM to 1 nM or less _D The amino acid sequence according to any one of aspects A-1 to A-25, which is capable of specifically binding to protein F of hRSV in (1).
Aspect A-27.10 ⁴ M ^-1 s ^-1 ~ About 10 ⁷ M ^-1 s ^-1 , Preferably 10 ⁵ M ^-1 s ^-1 -10 ⁷ M ^-1 s ^-1 , More preferably about 10 ⁶ M ^-1 s ^-1 More than k _on The amino acid sequence according to any one of aspects A-1 to A-26, which is capable of binding specifically to protein F of hRSV at a rate.
Aspect A-28.10 ^-2 s ^-1 (T _1/2 = 0.69 s) to 10 ^-4 s ^-1 (T _1/2 Is a few days), preferably 10 ^-3 s ^-1 -10 ^-4 s ^-1 The following k _off The amino acid sequence according to any one of aspects A-1 to A-27, which is capable of binding specifically to protein F of hRSV at a speed.
Aspect A-29. Neutralizing hRSV with an IC50 value of 100 nM to 1000 nM, preferably 100 nM to 500 nM or less, eg, as determined in a microneutralization assay for hRSV Long (eg, as described in Example 6). The amino acid sequence according to any one of Aspects A-1 to A-28, which can be used.
Aspect B-1. A Nanobody® having directionality and / or specifically binding to protein F of hRSV, wherein CDR2 comprises:
a) SEQ ID NO: 102,
b) a stretch of amino acid residues having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 102,
i) the amino acid residue stretch has aspartic acid (Asp, D) at position 6 (position 54 determined by Kabat numbering),
ii) the amino acid sequence comprising the amino acid residue stretch has the same, substantially the same or higher affinity as compared to the amino acid sequence comprising the amino acid residue stretch having no three, two or one amino acid difference ( The affinity is as measured by surface plasmon resonance) and binds to protein F of hRSV and / or the amino acid sequence comprising the amino acid residue stretch has three, two or one amino acid differences. Selected from amino acid residue stretches that have the same, approximately the same, or higher potency (as defined herein) as compared to the amino acid sequence comprising the amino acid residue stretch without Nanobodies® having directionality and / or specifically binding to protein F of hRSV.
Aspect B-2. CDR2 is
a) SEQ ID NO: 102, or
b) a stretch of amino acid residues having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 102,
i) the amino acid residue stretch has aspartic acid (Asp, D) at position 6 (position 54 determined by Kabat numbering),
ii) the amino acid sequence comprising the amino acid residue stretch has the same, substantially the same or higher affinity as compared to the amino acid sequence comprising the amino acid residue stretch having no three, two or one amino acid difference ( The affinity is as measured by surface plasmon resonance) and binds to protein F of hRSV and / or the amino acid sequence comprising the amino acid residue stretch has three, two or one amino acid differences. Selected from the group consisting of amino acid residue stretches that have the same, approximately the same, or higher potency (as defined herein) as compared to an amino acid sequence comprising the above amino acid residue stretches without And
At least one of CDR1 or CDR3 is
c) SEQ ID NO: 98;
d) An amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 98, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (as said affinity is measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference the amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Consisting of stretches of amino acid residues, which should have about the same or higher potency (as defined herein) Is selected from CDR1, and / or
e) SEQ ID NO: 121, and
f) an amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 121, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (such as measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference an amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Stretches of amino acid residues which should be of about the same or higher potency (as defined herein). CDR3 is selected from the group
The Nanobody (R) according to aspect B-1, selected from:
Aspect B-3. CDR1 is
a) SEQ ID NO: 98,
b) An amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 98, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (such as measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference an amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Consisting of stretches of amino acid residues, which should have about the same or higher potency (as defined herein) It is selected from,
CDR2 is
c) SEQ ID NO: 102,
d) a stretch of amino acid residues having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 102,
i) the amino acid residue stretch has aspartic acid (Asp, D) at position 6 (position 54 determined by Kabat numbering),
ii) the amino acid sequence comprising the amino acid residue stretch has the same, substantially the same or higher affinity as compared to the amino acid sequence comprising the amino acid residue stretch having no three, two or one amino acid difference ( The affinity is as measured by surface plasmon resonance) and binds to protein F of hRSV and / or the amino acid sequence comprising the amino acid residue stretch has three, two or one amino acid differences. Selected from the group consisting of amino acid residue stretches that have the same, approximately the same, or higher potency (as defined herein) as compared to an amino acid sequence comprising the above amino acid residue stretches without And
CDR3 is
e) SEQ ID NO: 121, and
f) an amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 121, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (such as measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference an amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Stretches of amino acid residues which should be of about the same or higher potency (as defined herein). It is selected from the group, Nanobody according to aspect B-1 or B-2 (R).
Aspect B-4. The Nanobody (registered trademark) according to any one of aspects B-1 to B-3, which comprises at least SEQ ID NO: 102.
Aspect B-5. At least SEQ ID NO: 102,
a) SEQ ID NO: 98,
b) an amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 98, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (as said affinity is measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference the amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; An amino acid residue stretch, which shall have about the same or higher potency (as defined herein);
c) SEQ ID NO: 121, and
d) An amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 121, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (as said affinity is measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference the amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Select from stretches of amino acid residues that should have about the same or higher potency (as defined herein). It is the, and at least one CDR sequence, according to embodiment B-4 Nanobodies.
Aspect B-6. At least SEQ ID NO: 102,
a) SEQ ID NO: 98, and
b) an amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 98, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (as said affinity is measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference the amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Select from stretches of amino acid residues that should have about the same or higher potency (as defined herein) CDR1 and sequences,
c) SEQ ID NO: 121, and
d) An amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 121, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (as said affinity is measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference the amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Select from stretches of amino acid residues that should have about the same or higher potency (as defined herein). Is the and a CDR3 sequence, Nanobody according to aspect B-4 or B-5 (R).
Aspect B-7. The Nanobody (R) according to any one of aspects B-4 to B-6, comprising at least SEQ ID NO: 102 and at least one CDR sequence selected from SEQ ID NO: 98 and SEQ ID NO: 121.
Aspect B-8. The Nanobody (registered trademark) according to any one of aspects B-4 to B-7, comprising SEQ ID NO: 98, SEQ ID NO: 102, and SEQ ID NO: 121.
Aspect B-9. The Nanobody (R) according to any one of aspects B-1 to B-8, which is in essentially isolated form.
Aspect B-10 Dissociation constant (K) of 0.1000 nM to 1 nM or less, preferably 100 nM to 1 nM or less, more preferably 10 nM to 1 nM or less _D The Nanobody (registered trademark) according to any one of aspects B-1 to B-9, which is capable of specifically binding to protein F of hRSV in (A).
Aspect B-11.10. ⁴ M ^-1 s ^-1 ~ About 10 ⁷ M ^-1 s ^-1 , Preferably 10 ⁵ M ^-1 s ^-1 -10 ⁷ M ^-1 s ^-1 , More preferably about 10 ⁶ M ^-1 s ^-1 More than k _on Nanobody according to any one of aspects B-1 to B-10, which is capable of binding specifically to protein F of hRSV at a rate.
Aspect B-12.10. ^-2 s ^-1 (T _1/2 = 0.69 s) to 10 ^-4 s ^-1 (T _1/2 Is a few days), preferably 10 ^-3 s ^-1 -10 ^-4 s ^-1 The following k _off Nanobody (R) according to any one of aspects B-1 to B-11, capable of binding specifically to protein F of hRSV at a rate.
Embodiment B-13. Neutralizing hRSV with an IC50 value of 100 nM to 1000 nM, preferably 100 nM to 500 nM or less, for example as determined in a microneutralization assay for hRSV Long (as described in Example 6). Nanobody (registered trademark) according to any one of aspects B-1 to B-12, which can be used.
Aspect B-14. Nanobody (s) according to any one of aspects B-1 to B-13, which is a natural Nanobody (R) (from any suitable species) or a synthetic or semi-synthetic Nanobody (R). .
Aspect B-15. V _HH Sequence, partially humanized V _HH Sequence, fully humanized V _HH The Nanobody (R) according to any one of aspects B-1 to B-14, which is a Nanobody (R) obtained by a technique such as sequence, camelized heavy chain variable domain, or affinity maturation.
Aspect B-16. Nanobody (registered trademark) according to any one of aspects B-1 to B-15, which is a partially humanized Nanobody (registered trademark).
Aspect B-17. Nanobody (registered trademark) according to any one of aspects B-1 to B-16, which is a fully humanized Nanobody (registered trademark).
Aspect B-18. can bind to protein F of hRSV (as defined herein);
i) a humanized variant of the amino acid sequence of SEQ ID NO: 5 (see Table A-1), and / or
ii) the amino acid sequence of SEQ ID NO: 5 (see Table A-1) and / or SEQ ID NO: 60-SEQ ID NO: 76, SEQ ID NO: 138-SEQ ID NO: 141, and SEQ ID NO: 146-SEQ ID NO: 157 (Table A-4) ) Has at least 80% amino acid identity (ignoring the amino acid residues forming the CDR sequence to determine the degree of amino acid identity) with at least one of the amino acid sequences of
iii) Preferably one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to Kabat numbering are international Selected from the characteristic residues referred to in Tables A-3 to A-8 of Publication 08/020079.
Nanobody (registered trademark) according to any one of aspects B-1 to B-17, consisting essentially of humanized Nanobody (registered trademark).
Aspect C-1:
a) SEQ ID NO: 60 to SEQ ID NO: 76
b) an amino acid sequence having only one amino acid difference from one of SEQ ID NO: 60 to SEQ ID NO: 76, preferably only two, more preferably only one;
i) the amino acid sequence has glutamine (Gln, Q) at position 105 (the position is determined by Kabat numbering);
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
An amino acid sequence having directionality and / or specifically binding to protein F of hRSV selected from the group consisting of:
Aspect C-2: Amino acid sequence according to Aspect C-1, comprising or consisting essentially of one of SEQ ID NO: 60 to SEQ ID NO: 76.
Aspect C-3:
a) SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76
b) only three, preferably only two and more preferably only one amino acid difference from one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75 and SEQ ID NO: 76 An amino acid sequence having
i) the amino acid sequence has glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and / or arginine (Arg, R) at position 83 (the above positions are determined by Kabat numbering; Desired),
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
An amino acid sequence having directionality and / or specifically binding to protein F of hRSV selected from the group consisting of:
Aspect C-4:
a) SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76
b) only three, preferably only two and more preferably only one amino acid difference from one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75 and SEQ ID NO: 76 An amino acid sequence having
i) the amino acid sequence has glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and arginine (Arg, R) at position 83 (the above positions are determined by Kabat numbering) ),
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
An amino acid sequence according to aspect C-3, which is selected from (hat)
Aspect C-5: Aspect C-3, comprising or consisting essentially of one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76. The amino acid sequence according to C-4.
Aspect C-6:
a) SEQ ID NO: 65 and SEQ ID NO: 76,
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NO: 65 and SEQ ID NO: 76,
i) the amino acid sequence is aspartic acid (Asp, D) at position 54, glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and / or arginine (Arg, R) at position 83; (The position is determined by Kabat numbering),
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
An amino acid sequence having directionality and / or specifically binding to protein F of hRSV selected from the group consisting of:
Aspect C-7:
a) SEQ ID NO: 65 and SEQ ID NO: 76,
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NO: 65 and SEQ ID NO: 76,
i) the amino acid sequence has aspartic acid (Asp, D) at position 54, glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and arginine (Arg, R) at position 83; (The above position is obtained by Kabat numbering)
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
An amino acid sequence according to aspect C-6, which is selected from:
Aspect C-8:
a) SEQ ID NO: 146 to SEQ ID NO: 153
b) an amino acid sequence having only one, preferably two, more preferably only one amino acid difference from one of SEQ ID NOs: 146 to 153,
i) the amino acid sequence has proline (Pro, P) at position 14, arginine (Arg, R) at position 19, leucine (Leu, L) at position 20, and leucine (Leu, L) at position 108 (The above position is determined by Kabat numbering),
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
An amino acid sequence having directionality and / or specifically binding to protein F of hRSV selected from the group consisting of:
Aspect C-9: Amino acid sequence according to Aspect C-8, comprising or consisting essentially of one of SEQ ID NO: 146 to SEQ ID NO: 153.
Aspect C-10:
a) SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153,
i) the amino acid sequence is proline (Pro, P) at position 14, arginine (Arg, R) at position 19, leucine (Leu, L) at position 20, and leucine (Leu, L) at position 108
And arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85, and / or glutamine (Gln, Q) at position 105 (the above positions are determined by Kabat numbering). ,
ii) the amino acid sequence has the same, substantially the same or higher affinity compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
An amino acid sequence having directionality and / or specifically binding to protein F of hRSV selected from the group consisting of:
Aspect C-11:
a) SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153,
i) the amino acid sequence is
SEQ ID NO: 146 (this amino acid sequence preferably has glutamine (Gln, Q) at position 105);
SEQ ID NO: 147 (this amino acid sequence preferably has arginine (Arg, R) at position 83 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 148 (this amino acid sequence preferably has glutamic acid (Glu, E) at position 85 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 149 (this amino acid sequence preferably has arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 151 (this amino acid sequence preferably has arginine (Arg, R) at position 83);
SEQ ID NO: 152 (this amino acid sequence preferably has glutamic acid (Glu, E) at position 85);
SEQ ID NO: 153 (this amino acid sequence preferably has arginine (Arg, R) at position 83 and glutamic acid (Glu, E) at position 85)
The amino acid sequence has a proline (Pro, P) at position 14 and an arginine (Arg, R) at position 19, as in the case of having only 3, preferably 2, and more preferably only 1 amino acid difference. Leucine (Leu, L) at position 20, Leucine (Leu, L) at position 108, arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85, and / or glutamine at position 105 (Gln, Q) (the position is determined by Kabat numbering),
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
An amino acid sequence according to aspect C-10, which is selected from:
Aspect C-12: Amino acid sequence according to Aspect C-10 or C-11, comprising or consisting essentially of one of SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153.
Aspect C-13: One or more amino acid residues are selected from a Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54D mutated sequence. An amino acid sequence comprising or consisting essentially of No. 5.
Aspect C-14: Two or more amino acid residues are selected from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and a sequence selected from Gly54D. The amino acid sequence according to aspect C-13, comprising or consisting essentially of 5.
Aspect C-15: Three or more amino acid residues are selected from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and a sequence selected from Gly54D. 5. The amino acid sequence according to aspect C-14, comprising or consisting essentially of 5.
Aspect C-16: Four or more amino acid residues are selected from the sequence of Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54D. 5. The amino acid sequence according to aspect C-15, comprising or consisting essentially of 5.
Aspect C-17: Five or more amino acid residues undergo sequence selected from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54D. The amino acid sequence according to aspect C-16, comprising or consisting essentially of 5.
Aspect C-18: Six or more amino acid residues are selected from the sequence of Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54D. 5. The amino acid sequence according to aspect C-17, comprising or consisting essentially of 5.
Aspect C-19: Seven or more amino acid residues are selected from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and a sequence selected from Gly54D. The amino acid sequence according to aspect C-18, comprising or consisting essentially of 5.
Aspect C-20: Eight or more amino acid residues are selected from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and a sequence selected from Gly54D. The amino acid sequence according to aspect C-19, comprising or consisting essentially of 5.
Aspect C-21: Nine or more amino acid residues are selected from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and a sequence selected from the mutations Gly54D. The amino acid sequence according to aspect C-20, comprising or consisting essentially of 5.
Aspect C-22: Ten or more amino acid residues are selected from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and a sequence selected from Gly54D. 5. The amino acid sequence according to aspect C-21, comprising or consisting essentially of 5.
Aspect C-23: wherein the amino acid residue has a mutation from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, or a sequence from SEQ ID NO: 5, or a mutation from Gly54D. An amino acid sequence according to aspect C-22 consisting essentially of.
Aspect C-24: One or more amino acid residues comprising or from SEQ ID NO: 5, having a mutation selected from Ser19R, Ile20Leu, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54D. An amino acid sequence according to aspect C-13, consisting essentially of.
Aspect C-25: An embodiment comprising or consisting essentially of SEQ ID NO: 5, wherein the one or more amino acid residues is mutated selected from Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54D. The amino acid sequence according to C-13.
Aspect C-26: Amino acid sequence according to aspect C-25, wherein the amino acid residue comprises or consists essentially of SEQ ID NO: 5, mutated Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54D. .
Aspect C-27: Amino acid sequence comprising or consisting essentially of SEQ ID NO: 5, wherein one or more amino acid residues is mutated selected from Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu.
Aspect C-28: comprises or consists essentially of SEQ ID NO: 5, wherein one or more amino acid residues is mutated selected from Ala14Pro, Ser19Arg, Ile20Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu. Amino acid sequence
Aspect C-29:
The amino acid residue is
Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Gly78Leu, Ala83Arg, Asp85Glu, and Arg105Gln,
Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54Asp,
Gly54Asp,
Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Ala83Arg,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Asp85Glu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Arg105Gln,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Asp85Glu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Arg105Gln,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Asp85Glu, and Arg105Gln, or
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, Asp85Glu, and Arg105Gln
The amino acid sequence according to aspect C-8, comprising or consisting essentially of SEQ ID NO: 5 mutated.
Aspect C-30: Amino acid sequence comprising or consisting essentially of SEQ ID NO: 62.
Aspect C-31: An amino acid sequence comprising or consisting essentially of SEQ ID NO: 65.
Aspect C-32: Amino acid sequence comprising or consisting essentially of SEQ ID NO: 76.
Aspect C-33: Amino acid sequence comprising or consisting essentially of any of SEQ ID NO: 146 to SEQ ID NO: 153.
Embodiment C-34: Dissociation constant (K) of 1000 nM to 1 nM or less, preferably 100 nM to 1 nM or less, more preferably 10 nM to 1 nM or less _D The amino acid sequence according to any one of aspects C-1 to C-33, wherein the amino acid sequence can specifically bind to protein F of hRSV in (1).
Aspect C-35: 10 ⁴ M ^-1 s ^-1 ~ About 10 ⁷ M ^-1 s ^-1 , Preferably 10 ⁵ M ^-1 s ^-1 -10 ⁷ M ^-1 s ^-1 , More preferably about 10 ⁶ M ^-1 s ^-1 More than k _on The amino acid sequence according to any one of aspects C-1 to C-34, which is capable of binding specifically to protein F of hRSV at a rate.
Aspect C-36: 10 ^-2 s ^-1 (T _1/2 = 0.69 s) to 10 ^-4 s ^-1 (T _1/2 Is a few days), preferably 10 ^-3 s ^-1 -10 ^-4 s ^-1 The following k _off The amino acid sequence according to any one of aspects C-1 to C-35, which is capable of binding specifically to protein F of hRSV at a rate.
Aspect C-37: Neutralizing hRSV with an IC50 value of 100 nM to 1000 nM, preferably 100 nM to 500 nM or less, eg, as determined in a microneutralization assay for hRSV Long (eg, as described in Example 6). An amino acid sequence according to any one of aspects C-1 to C-36, which can be
Aspect C-38: of the aspect C-1 to C-37, which specifically binds to antigenic site II on protein F of hRSV and / or competes with SYNAGIS® for binding of protein F of hRSV. An amino acid sequence according to any one of the above.
Aspect D-1:
a) SEQ ID NO: 60 to SEQ ID NO: 76
b) an amino acid sequence having only one amino acid difference from one of SEQ ID NO: 60 to SEQ ID NO: 76, preferably only two, more preferably only one;
i) the amino acid sequence has glutamine (Gln, Q) at position 105 (the position is determined by Kabat numbering);
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
A Nanobody® having directionality and / or specifically binding to protein F of hRSV selected from the group consisting of:
Aspect D-2: A Nanobody according to Aspect D-1, comprising or consisting essentially of one of SEQ ID NO: 60-76.
Aspect D-3:
a) SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76
b) only three, preferably only two and more preferably only one amino acid difference from one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75 and SEQ ID NO: 76 An amino acid sequence having
i) the amino acid sequence has glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and / or arginine (Arg, R) at position 83 (the above positions are determined by Kabat numbering; Desired),
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
A Nanobody® having directionality and / or specifically binding to protein F of hRSV selected from the group consisting of:
Aspect D-4:
a) SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76
b) only three, preferably only two and more preferably only one amino acid difference from one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75 and SEQ ID NO: 76 An amino acid sequence having
i) the amino acid sequence has glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and arginine (Arg, R) at position 83 (the above positions are determined by Kabat numbering) ),
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
The Nanobody according to aspect D-3, wherein the Nanobody is selected from:
Aspect D-5: Aspect D-3, comprising or consisting essentially of one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76. Nanobody (registered trademark) according to D-4.
Aspect D-6:
a) SEQ ID NO: 65 and SEQ ID NO: 76,
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NO: 65 and SEQ ID NO: 76,
i) the amino acid sequence is aspartic acid (Asp, D) at position 54, glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and / or arginine (Arg, R) at position 83; (The position is determined by Kabat numbering),
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
A Nanobody® having directionality and / or specifically binding to protein F of hRSV selected from the group consisting of:
Aspect D-7:
a) SEQ ID NO: 65 and SEQ ID NO: 76,
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NO: 65 and SEQ ID NO: 76,
i) the amino acid sequence has aspartic acid (Asp, D) at position 54, glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and arginine (Arg, R) at position 83; (The position is determined by Kabat numbering), and the amino acid sequence has the same, almost the same or higher affinity (amino acid sequence) compared to an amino acid sequence having no three, two or one amino acid difference. Sex is as measured by surface plasmon resonance) and binds to protein F of hRSV and / or compares the amino acid sequence with an amino acid sequence that does not have three, two or one amino acid difference. Amino acid sequences that have the same, about the same, or higher potency (as defined herein)
The Nanobody (R) according to aspect D-6, selected from:
Aspect D-8:
a) SEQ ID NO: 146 to SEQ ID NO: 153
b) an amino acid sequence having only one, preferably two, more preferably only one amino acid difference from one of SEQ ID NOs: 146 to 153,
i) the amino acid sequence has proline (Pro, P) at position 14, arginine (Arg, R) at position 19, leucine (Leu, L) at position 20, and leucine (Leu, L) at position 108 (The above position is determined by Kabat numbering),
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
A Nanobody® having directionality and / or specifically binding to protein F of hRSV selected from the group consisting of:
Aspect D-9: Nanobody according to aspect D-8, comprising or consisting essentially of one of SEQ ID NOs: 146-153.
Aspect D-10:
a) SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153,
i) the amino acid sequence comprises proline (Pro, P) at position 14, arginine (Arg, R) at position 19, leucine (Leu, L) at position 20, and leucine (Leu, L) at position 108; Arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85, and / or glutamine (Gln, Q) at position 105 (the position is determined by Kabat numbering);
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
A Nanobody that is directional and / or specifically binds to hRSV protein F selected from:
Aspect D-11:
a) SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153,
i) the amino acid sequence is
SEQ ID NO: 146 (this amino acid sequence preferably has glutamine (Gln, Q) at position 105); SEQ ID NO: 147 (This amino acid sequence preferably has arginine (Arg, R) at position 83 and glutamine (Gln, Q)),
SEQ ID NO: 148 (this amino acid sequence preferably has glutamic acid (Glu, E) at position 85 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 149 (this amino acid sequence preferably has arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 151 (this amino acid sequence preferably has arginine (Arg, R) at position 83);
SEQ ID NO: 152 (this amino acid sequence preferably has glutamic acid (Glu, E) at position 85);
SEQ ID NO: 153 (this amino acid sequence preferably has arginine (Arg, R) at position 83 and glutamic acid (Glu, E) at position 85)
The amino acid sequence has a proline (Pro, P) at position 14 and an arginine (Arg, R) at position 19, as in the case of having only 3, preferably 2, and more preferably only 1 amino acid difference. Leucine (Leu, L) at position 20, Leucine (Leu, L) at position 108, arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85, and / or glutamine at position 105 (Gln, Q) (the position is determined by Kabat numbering),
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
The nanobody according to aspect D-10, wherein the nanobody is selected from:
Aspect D-12: Nanobody according to aspect D-10 or D-11, comprising or consisting essentially of one of SEQ ID NOs: 146-149 and SEQ ID NOs: 151-153.
Aspect D-13: One or more amino acid residues are selected from a Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54D mutated sequence. A Nanobody comprising or consisting essentially of No. 5.
Aspect D-14: Two or more amino acid residues are selected from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and a sequence selected from Gly54D. The Nanobody (R) according to aspect D-13, comprising or consisting essentially of 5.
Aspect D-15: Three or more amino acid residues are selected from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and a sequence selected from Gly54D. Nanobody (R) according to aspect D-14, comprising or consisting essentially of 5.
Aspect D-16: Four or more amino acid residues are selected from the sequence of Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54D. The Nanobody (R) according to aspect D-150, comprising or consisting essentially of 5.
Aspect D-17: Five or more amino acid residues are selected from the sequence of Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54D. The Nanobody (R) according to aspect D-16, comprising or consisting essentially of 5.
Aspect D-18: Six or more amino acid residues are selected from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and a sequence selected from Gly54D. 5. The Nanobody (R) according to aspect D-17, comprising or consisting essentially of 5.
Aspect D-19: Seven or more amino acid residues are selected from the sequence of Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54D. Nanobody (R) according to aspect D-18, comprising or consisting essentially of 5.
Aspect D-20: Eight or more amino acid residues are selected from the sequence of Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54D. Nanobody (R) according to aspect D-19, comprising or consisting essentially of 5.
Aspect D-21: Nine or more amino acid residues are selected from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and a mutation selected from Gly54D. Nanobody (R) according to aspect D-20, comprising or consisting essentially of 5.
Aspect D-22: 10 or more amino acid residues are selected from the sequence of Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54D. The Nanobody (R) according to aspect D-21, comprising or consisting essentially of 5.
Aspect D-23: Amino acid residue mutated from or including the sequence of Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54D. The Nanobody (R) according to embodiment D-22, consisting essentially of:
Aspect D-24: One or more amino acid residues comprising or from SEQ ID NO: 5, having a mutation selected from Ser19R, Ile20Leu, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54D. The Nanobody® according to aspect D-13, consisting essentially of:
Aspect D-25: An embodiment comprising or consisting essentially of SEQ ID NO: 5, wherein one or more amino acid residues is mutated selected from Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54D. Nanobody (registered trademark) according to D-8.
Aspect D-26: Nanobody according to aspect D-20, wherein the amino acid residue comprises or consists essentially of SEQ ID NO: 5, wherein the amino acid residue is mutated Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54D. Registered trademark).
Aspect D-27: A Nanobody comprising or consisting essentially of SEQ ID NO: 5, wherein one or more amino acid residues is mutated selected from Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu. ).
Aspect D-28: comprises or consists essentially of SEQ ID NO: 5, wherein one or more amino acid residues is mutated selected from Ala14Pro, Ser19Arg, Ile20Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu. Nanobody (registered trademark).
Aspect D-29:
The amino acid residue is
Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Gly78Leu, Ala83Arg, Asp85Glu, and Arg105Gln,
Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54Asp,
Gly54Asp,
Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Ala83Arg,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Asp85Glu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Arg105Gln,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Asp85Glu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Arg105Gln,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Asp85Glu, and Arg105Gln, or
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, Asp85Glu, and Arg105Gln
The Nanobody (R) according to aspect D-13, comprising or consisting essentially of SEQ ID NO: 5 mutated.
Aspect D-30: A Nanobody comprising or consisting essentially of SEQ ID NO: 62.
Aspect D-31: A Nanobody comprising or consisting essentially of SEQ ID NO: 65.
Aspect D-32: A Nanobody comprising or consisting essentially of SEQ ID NO: 76.
Aspect D-33: Nanobody® comprising or consisting essentially of any of SEQ ID NOs: 146-153.
Aspect D-34: Dissociation constant (K) of 1000 nM to 1 nM, preferably 100 nM to 1 nM, more preferably 10 nM to 1 nM _D The Nanobody (registered trademark) according to any one of aspects D-1 to D-33, which is capable of specifically binding to protein F of hRSV in the above (1).
Aspect D-35: 10 ⁴ M ^-1 s ^-1 ~ About 10 ⁷ M ^-1 s ^-1 , Preferably 10 ⁵ M ^-1 s ^-1 -10 ⁷ M ^-1 s ^-1 , More preferably about 10 ⁶ M ^-1 s ^-1 More than k _on Nanobody (R) according to any one of aspects D-1 to D-34, which is capable of binding specifically to protein F of hRSV at a rate.
Aspect D-36: 10 ^-2 s ^-1 (T _1/2 = 0.69 s) to 10 ^-4 s ^-1 (T _1/2 Is a few days), preferably 10 ^-3 s ^-1 -10 ^-4 s ^-1 The following k _off Nanobody <(R)> according to any one of aspects D-1 to D-35, which is capable of binding specifically to protein F of hRSV at a rate.
Aspect D-37: Neutralizing hRSV with an IC50 value of 100 nM to 1000 nM, preferably 100 nM to 500 nM or less, eg, as determined in a microneutralization assay for hRSV Long (eg, as described in Example 6). Nanobody (registered trademark) according to any one of aspects D-1 to D-36, which is capable of doing so.
Aspect D-38: Of the aspects D-1 to D-37, which specifically bind to antigenic site II on protein F of hRSV and / or compete with Synagis® for binding of hRSV to protein F. Nanobody (registered trademark) according to any one of the above.
Aspect W-1: has directivity to and / or specifically binds to protein F of hRSV, and the amino acid sequence has an aspartic acid (Asp, D) at position 1 (the position is determined by Kabat numbering). The amino acid sequence according to any one of aspects A-1 to A-29 and C-1 to C-38, wherein
Aspect W-2:
a) SEQ ID NO: 138 to SEQ ID NO: 141 and SEQ ID NO: 154 to SEQ ID NO: 157;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 138 to 141 and 154 to 157,
i) the amino acid sequence has aspartic acid (Asp, D) at position 1 (the position is determined by Kabat numbering);
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
An amino acid sequence having directionality and / or specifically binding to protein F of hRSV selected from the group consisting of:
Aspect W-3: Amino acid sequence according to aspect W-2, comprising or consisting essentially of one of SEQ ID NOs: 138-141 and SEQ ID NOs: 154-157.
Aspect W-4. Amino acid sequence comprising or consisting essentially of SEQ ID NO: 5, wherein the glutamic acid at position 1 has been changed to aspartic acid.
Aspect W-5. An amino acid sequence comprising or consisting essentially of SEQ ID NO: 62, wherein the glutamic acid at position 1 is changed to aspartic acid.
Aspect W-6. Amino acid sequence comprising or consisting essentially of SEQ ID NO: 65, wherein the glutamic acid at position 1 has been changed to aspartic acid.
Aspect W-7. An amino acid sequence comprising or consisting essentially of SEQ ID NO: 76, wherein glutamic acid at position 1 has been changed to aspartic acid.
Aspect W-8. Amino acid sequence comprising or consisting essentially of SEQ ID NO: 75, wherein glutamic acid at position 1 has been changed to aspartic acid.
Aspect W-9. Amino acid sequence comprising or consisting essentially of SEQ ID NO: 147, wherein glutamic acid at position 1 is changed to aspartic acid.
Aspect W-10. Amino acid sequence comprising or consisting essentially of SEQ ID NO: 149, wherein glutamic acid at position 1 is changed to aspartic acid.
Aspect W-11 An amino acid sequence comprising or consisting essentially of SEQ ID NO: 153, wherein glutamic acid at position 1 is changed to aspartic acid.
Aspect W-12: One or more amino acid residues are selected from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54D. An amino acid sequence comprising or consisting essentially of SEQ ID NO: 5, wherein the glutamic acid at position 1 has been changed to aspartic acid.
Aspect W-13: Two or more amino acid residues are selected from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54D. The amino acid sequence of embodiment W-12, wherein the amino acid sequence comprises or consists essentially of SEQ ID NO: 5, wherein the glutamic acid at position is changed to aspartic acid.
Aspect W-14: Three or more amino acid residues are selected from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54D. The amino acid sequence according to embodiment W-13, wherein the amino acid sequence comprises or consists essentially of SEQ ID NO: 5, wherein the glutamic acid at position is changed to aspartic acid.
Aspect W-15: Four or more amino acid residues are selected from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54D. The amino acid sequence according to embodiment W-14, wherein the amino acid sequence comprises or consists essentially of SEQ ID NO: 5, wherein the glutamic acid at position is changed to aspartic acid.
Aspect W-16: Five or more amino acid residues are selected from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54D. The amino acid sequence of embodiment W-15, wherein the amino acid sequence comprises or consists essentially of SEQ ID NO: 5, wherein the glutamic acid at position is changed to aspartic acid.
Aspect W-17: Six or more amino acid residues are selected from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54D. The amino acid sequence of embodiment W-16, wherein the amino acid sequence comprises or consists essentially of SEQ ID NO: 5, wherein the glutamic acid at position is changed to aspartic acid.
Aspect W-18: Seven or more amino acid residues are selected from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54D. The amino acid sequence of embodiment W-17, wherein the amino acid sequence comprises or consists essentially of SEQ ID NO: 5, wherein the glutamic acid at position is changed to aspartic acid.
Aspect W-19: Eight or more amino acid residues are selected from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54D. The amino acid sequence of embodiment W-18, wherein the amino acid sequence comprises or consists essentially of SEQ ID NO: 5, wherein the glutamic acid at position is changed to aspartic acid.
Aspect W-20: Nine or more amino acid residues are selected from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54D. The amino acid sequence of embodiment W-19, wherein the amino acid sequence comprises or consists essentially of SEQ ID NO: 5, wherein the glutamic acid at position is changed to aspartic acid.
Embodiment W-21: Ten or more amino acid residues are selected from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54D and are mutated. The amino acid sequence of embodiment W-20, wherein the amino acid sequence comprises or consists essentially of SEQ ID NO: 5, wherein the glutamic acid at position is changed to aspartic acid.
Aspect W-22: Amino acid residue has been mutated at amino acid position glutamic acid with amino acid residue Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54D. The amino acid sequence of embodiment W-21, wherein the amino acid sequence comprises or consists essentially of SEQ ID NO: 5, which is altered.
Aspect W-23: One or more amino acid residues have undergone a mutation selected from Ser19R, Ile20Leu, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54D, wherein glutamic acid at position 1 is aspartic acid. The amino acid sequence of embodiment W-12, wherein the amino acid sequence comprises or consists essentially of SEQ ID NO: 5, which is altered.
Aspect W-24: A sequence wherein one or more amino acid residues has undergone a mutation selected from Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54D, wherein glutamic acid at position 1 has been changed to aspartic acid. The amino acid sequence according to embodiment W-12, comprising or consisting essentially of No. 5.
Aspect W-25: SEQ ID NO: 5, wherein the amino acid residue has been mutated Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54D and glutamic acid at position 1 has been changed to aspartic acid. Amino acid sequence according to aspect W-24, consisting essentially of:
Aspect W-26: SEQ ID NO: 5 wherein one or more amino acid residues has undergone a mutation selected from Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu, and wherein glutamic acid at position 1 has been changed to aspartic acid. An amino acid sequence comprising or consisting essentially of
Aspect W-27: One or more amino acid residues has undergone a mutation selected from Ala14Pro, Ser19Arg, Ile20Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu, wherein glutamic acid at position 1 has been changed to aspartic acid. An amino acid sequence comprising or consisting essentially of SEQ ID NO: 5.
Aspect W-28:
The amino acid residue is
Glu1Asp, Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Glu1Asp, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Glu1Asp, Gly78Leu, Ala83Arg, Asp85Glu, and Arg105Gln;
Glu1Asp, Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Glu1Asp, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Glu1Asp, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54Asp,
Glu1Asp and Gly54Asp, Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Ala83Arg;
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Asp85Glu,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Arg105Gln,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Asp85Glu,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Arg105Gln,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Asp85Glu, and Arg105Gln, or
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, Asp85Glu, and Arg105Gln
The amino acid sequence according to aspects W-2 or W-3, comprising or consisting essentially of SEQ ID NO: 5 mutated.
Aspect W-29: Amino acid sequence comprising or consisting essentially of SEQ ID NO: 138.
Aspect W-30: Amino acid sequence comprising or consisting essentially of SEQ ID NO: 139.
Aspect W-31: An amino acid sequence comprising or consisting essentially of SEQ ID NO: 140.
Aspect W-32: Amino acid sequence comprising or consisting essentially of SEQ ID NO: 141.
Aspect W-33: Amino acid sequence comprising or consisting essentially of any of SEQ ID NOs: 154-157.
Embodiment W-34: Dissociation constant (K) of 1000 nM to 1 nM or less, preferably 100 nM to 1 nM or less, more preferably 10 nM to 1 nM or less _D The amino acid sequence according to any one of aspects W-1 to W-33, which is capable of specifically binding to protein F of hRSV in (1).
Aspect W-35: 10 ⁴ M ^-1 s ^-1 ~ About 10 ⁷ M ^-1 s ^-1 , Preferably 10 ⁵ M ^-1 s ^-1 -10 ⁷ M ^-1 s ^-1 , More preferably about 10 ⁶ M ^-1 s ^-1 More than k _on The amino acid sequence according to any one of aspects W-1 to W-34, which is capable of binding specifically to protein F of hRSV at a rate.
Aspect W-36: 10 ^-2 s ^-1 (T _1/2 = 0.69 s) to 10 ^-4 s ^-1 (T _1/2 Is a few days), preferably 10 ^-3 s ^-1 -10 ^-4 s ^-1 The following k _off The amino acid sequence according to any one of aspects W-1 to W-35, which is capable of binding specifically to protein F of hRSV at a rate.
Aspect W-37: Neutralizing hRSV with an IC50 value of 100 nM to 1000 nM, preferably 100 nM to 500 nM or less, eg, as determined in a microneutralization assay for hRSV Long (eg, as described in Example 6). An amino acid sequence according to any one of aspects W-1 to W-36, which can be
Aspect W-38: of the aspect W-1 to W-37, which specifically binds to antigenic site II on protein F of hRSV and / or competes with SYNAGIS® for binding of protein F of hRSV. An amino acid sequence according to any one of the above.
Aspect X-1 Any one of Aspects B-1 to B-18 and Aspects D-1 to D-38 having aspartic acid (Asp, D) at position 1 (the position is determined by Kabat numbering) 4. Nanobodies having directivity and / or specifically binding to protein F of hRSV according to any one of the above.
Aspect X-2:
a) SEQ ID NO: 138 to SEQ ID NO: 141 and SEQ ID NO: 154 to SEQ ID NO: 157;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 138 to 141 and 154 to 157,
i) the amino acid sequence has aspartic acid (Asp, D) at position 1 (the position is determined by Kabat numbering);
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
A Nanobody® having directionality and / or specifically binding to protein F of hRSV selected from the group consisting of:
Aspect X-3: Nanobody according to aspect X-2, comprising or consisting essentially of one of SEQ ID NOs: 138-141 and SEQ ID NOs: 154-157.
Aspect X-4. A Nanobody comprising or consisting essentially of SEQ ID NO: 5, wherein the glutamic acid at position 1 has been changed to aspartic acid.
Aspect X-5. A Nanobody comprising or consisting essentially of SEQ ID NO: 62, wherein the glutamic acid at position 1 has been changed to aspartic acid.
Aspect X-6. A Nanobody comprising or consisting essentially of SEQ ID NO: 65, wherein the glutamic acid at position 1 is changed to aspartic acid.
Aspect X-7. A Nanobody comprising or consisting essentially of SEQ ID NO: 76, wherein the glutamic acid at position 1 has been changed to aspartic acid.
Aspect X-8. A Nanobody comprising or consisting essentially of SEQ ID NO: 75, wherein the glutamic acid at position 1 has been changed to aspartic acid.
Aspect X-9. A Nanobody comprising or consisting essentially of SEQ ID NO: 147, wherein the glutamic acid at position 1 is changed to aspartic acid.
Aspect X-10. A Nanobody comprising or consisting essentially of SEQ ID NO: 149, wherein the glutamic acid at position 1 is changed to aspartic acid.
Aspect X-11. A Nanobody comprising or consisting essentially of SEQ ID NO: 153, wherein the glutamic acid at position 1 is changed to aspartic acid.
Aspect X-12: One or more amino acid residues are selected from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54D. A Nanobody comprising or consisting essentially of SEQ ID NO: 5, wherein the glutamic acid at position 1 has been changed to aspartic acid.
Aspect X-13: Two or more amino acid residues are selected from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54D. The Nanobody <(R)> according to embodiment X-12, comprising or consisting essentially of SEQ ID NO: 5, wherein the glutamic acid at position is changed to aspartic acid.
Aspect X-14: Three or more amino acid residues are selected from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54D. The Nanobody (R) according to embodiment X-13, comprising or consisting essentially of SEQ ID NO: 5, wherein the glutamic acid at position is changed to aspartic acid.
Aspect X-15: Four or more amino acid residues are selected from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54D. The Nanobody (R) according to embodiment X-14, comprising or consisting essentially of SEQ ID NO: 5, wherein the glutamic acid at position is changed to aspartic acid.
Aspect X-16: Five or more amino acid residues are selected from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54D. The Nanobody (R) according to embodiment X-15, comprising or consisting essentially of SEQ ID NO: 5, wherein the glutamic acid at position is changed to aspartic acid.
Aspect X-17: Six or more amino acid residues are selected from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54D. The Nanobody (R) according to embodiment X-16, comprising or consisting essentially of SEQ ID NO: 5, wherein the glutamic acid at position is changed to aspartic acid.
Aspect X-18: Seven or more amino acid residues have been selected from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54D. The Nanobody according to aspect X-17, comprising or consisting essentially of SEQ ID NO: 5, wherein the glutamic acid at position is changed to aspartic acid.
Aspect X-19: Eight or more amino acid residues are selected from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54D. The Nanobody (R) according to embodiment X-18, comprising or consisting essentially of SEQ ID NO: 5, wherein the glutamic acid at position is changed to aspartic acid.
Aspect X-20: Nine or more amino acid residues are selected from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu and Gly54D. The Nanobody <(R)> according to embodiment X-19, comprising or consisting essentially of SEQ ID NO: 5, wherein the glutamic acid at position is changed to aspartic acid.
Aspect X-21: Ten or more amino acid residues are selected from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54D and are mutated. The Nanobody® according to aspect X-20, comprising or consisting essentially of SEQ ID NO: 5, wherein the glutamic acid at position is changed to aspartic acid.
Aspect X-22: Amino acid residue has been mutated to amino acid at position Gal at amino acid Gal at amino acid position Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54D. The Nanobody <(R)> according to aspect X-21, which is altered, comprising or consists essentially of SEQ ID NO: 5.
Aspect X-23: One or more amino acid residues have undergone a mutation selected from Ser19R, Ile20Leu, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54D, wherein glutamic acid at position 1 is aspartic acid. The Nanobody according to embodiment X-12, which is altered, comprises or consists essentially of SEQ ID NO: 5.
Aspect X-24: A sequence wherein one or more amino acid residues has undergone a mutation selected from Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54D, wherein glutamic acid at position 1 has been changed to aspartic acid. The Nanobody® according to aspect X-12, comprising or consisting essentially of No. 5.
Aspect X-25 comprising or from SEQ ID NO: 5, wherein the amino acid residue has been mutated for Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54D and glutamic acid at position 1 has been changed to aspartic acid. The Nanobody (R) according to embodiment X-24, consisting essentially of:
Aspect X-26: SEQ ID NO: 5 wherein one or more amino acid residues has undergone a mutation selected from Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu, wherein glutamic acid at position 1 has been changed to aspartic acid. Or consisting essentially of Nanobody®.
Aspect X-27: One or more amino acid residues have undergone a mutation selected from Ala14Pro, Ser19Arg, Ile20Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu, wherein glutamic acid at position 1 has been changed to aspartic acid. A Nanobody® comprising or consisting essentially of SEQ ID NO: 5.
Aspect X-28:
The amino acid residue is
Glu1Asp, Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Glu1Asp, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Glu1Asp, Gly78Leu, Ala83Arg, Asp85Glu, and Arg105Gln;
Glu1Asp, Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Glu1Asp, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Glu1Asp, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54Asp,
Glu1Asp and Gly54Asp,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Ala83Arg;
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Asp85Glu,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Arg105Gln,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Asp85Glu,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Arg105Gln,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Asp85Glu, and Arg105Gln, or
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, Asp85Glu, and Arg105Gln
The Nanobody (R) according to aspect X-2, comprising or consisting essentially of SEQ ID NO: 5, mutated.
Aspect X-29: A Nanobody comprising or consisting essentially of SEQ ID NO: 138.
Aspect X-30: A Nanobody comprising or consisting essentially of SEQ ID NO: 139.
Aspect X-31: A Nanobody comprising or consisting essentially of SEQ ID NO: 140.
Aspect X-32: A Nanobody comprising or consisting essentially of SEQ ID NO: 141.
Aspect X-33: A Nanobody comprising or consisting essentially of any of SEQ ID NOs: 154-157.
Aspect X-34: Dissociation constant (K of 1000 nM to 1 nM or less, preferably 100 nM to 1 nM or less, more preferably 10 nM to 1 nM or less _D The Nanobody (registered trademark) according to any one of aspects X-1 to X-33, which is capable of specifically binding to protein F of hRSV in (1).
Aspect X-35: 10 ⁴ M ^-1 s ^-1 ~ About 10 ⁷ M ^-1 s ^-1 , Preferably 10 ⁵ M ^-1 s ^-1 -10 ⁷ M ^-1 s ^-1 , More preferably about 10 ⁶ M ^-1 s ^-1 More than k _on The Nanobody (R) according to any one of aspects X-1 to X-34, which is capable of binding specifically to protein F of hRSV at a rate.
Aspect X-36: 10 ^-2 s ^-1 (T _1/2 = 0.69 s) to 10 ^-4 s ^-1 (T _1/2 Is a few days), preferably 10 ^-3 s ^-1 -10 ^-4 s ^-1 The following k _off The Nanobody (R) according to any one of aspects X-1 to X-35, which is capable of binding specifically to protein F of hRSV at a rate.
Embodiment X-37: Neutralizing hRSV with an IC50 value of 100 nM to 1000 nM, preferably 100 nM to 500 nM or less, eg, as determined in a microneutralization assay for hRSV Long (eg, as described in Example 6). Nanobody (registered trademark) according to any one of aspects X-1 to X-36, which can be used.
Aspect X-38: of aspects X-I to X-37, which specifically binds to antigenic site II on protein F of hRSV and / or competes with SYNAGIS® for binding of protein F of hRSV. Nanobody (registered trademark) according to any one of the above.
Aspect E-1: One or more amino acid sequences according to any one of Aspects A-1 to A-29, Aspects C-1 to C-38, and Aspects W-1 to W-38, and / or Or comprising one or more Nanobodies (registered trademark) according to any one of aspects B-1 to B-18, aspects D-1 to D-38, and aspects X-1 to X-38, Or consisting essentially of, and optionally further comprising, one or more other amino acid binding units optionally linked via one or more peptide linkers.
Aspect E-2: Polypeptide according to aspect E-1, wherein said one or more other binding units is an immunoglobulin sequence.
Aspect E-3: wherein one or more of the other binding units is a domain antibody, an amino acid sequence suitable for use as a domain antibody, a single domain antibody, an amino acid sequence suitable for use as a single domain antibody, The polypeptide according to aspect E-1 or E-2, wherein the polypeptide is selected from the group consisting of "dAb", an amino acid sequence suitable for use as a dAb, or Nanobody (R).
Aspect E-4: The polypeptide according to any one of aspects E-1 to E-3, wherein said one or more amino acid sequences are immunoglobulin sequences.
Aspect E-5: One or more of the above amino acid sequences is a domain antibody, an amino acid sequence suitable for use as a domain antibody, a single domain antibody, an amino acid sequence suitable for use as a single domain antibody, “dAb The polypeptide according to any one of aspects E-1 to E-4, wherein the polypeptide is selected from the group consisting of: an amino acid sequence suitable for use as a dAb, or Nanobody®.
Aspect E-6: One or more Nanobodies (R) according to any one of Aspects B-1 to B-18, Aspects D-1 to D-8, and Aspects X-1 to X-38 The polypeptide according to any one of aspects E-1 to E-5, wherein the polypeptide comprises or consists essentially of one or more of the other binding units described above is a Nanobody®.
Aspect E-7: The polypeptide according to any one of aspects E-1 to E-6, which is a multivalent construct.
Aspect E-8: At least two amino acid sequences according to any one of Aspects A-1 to A-29 and Aspects C-1 to C-38, and / or Aspects B-1 to B-18 and Aspects Nanobody (registered trademark) according to any one of D-1 to D-38, and / or at least one amino acid sequence according to any one of embodiments W-1 to W-38, and / or an embodiment The multivalent polypeptide according to aspect E-7, comprising or consisting essentially of at least one Nanobody® according to any one of X-1 to X-38.
Aspect E-9: Multivalent polypeptide according to aspect E-8, wherein the at least two amino acid sequences and / or Nanobodies® are identical.
Aspect E-10:
a) SEQ ID NO: 102,
b) a stretch of amino acid residues having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 102,
i) the amino acid residue stretch has aspartic acid (Asp, D) at position 6 (position 54 determined by Kabat numbering),
ii) the amino acid sequence comprising the amino acid residue stretch has the same, substantially the same or higher affinity as compared to the amino acid sequence comprising the amino acid residue stretch having no three, two or one amino acid difference ( The affinity is as measured by surface plasmon resonance) and binds to protein F of hRSV and / or the amino acid sequence comprising the amino acid residue stretch has three, two or one amino acid differences. An amino acid selected from an amino acid residue stretch that has the same, approximately the same, or a higher potency (as defined herein) as compared to an amino acid sequence comprising the above amino acid residue stretch without Whether it comprises at least two amino acid sequences and / or Nanobodies®, including at least one residue stretch Or which consists essentially of, multivalent polypeptide according to any of aspects E-7~E-9.
Aspect E-11:
a) SEQ ID NO: 102,
b) a stretch of amino acid residues having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 102,
i) the amino acid residue stretch has aspartic acid (Asp, D) at position 6 (position 54 determined by Kabat numbering),
ii) the amino acid sequence comprising the amino acid residue stretch has the same, substantially the same or higher affinity as compared to the amino acid sequence comprising the amino acid residue stretch having no three, two or one amino acid difference ( The affinity is as measured by surface plasmon resonance) and binds to protein F of hRSV and / or the amino acid sequence comprising said stretch of amino acid residues differs by three, two or one amino acid An amino acid selected from an amino acid residue stretch that has the same, approximately the same, or a higher potency (as defined herein) as compared to an amino acid sequence comprising the above amino acid residue stretch without Including at least one residue stretch,
At least one stretch,
c) SEQ ID NO: 98;
d) An amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 98, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (as said affinity is measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference the amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; An amino acid residue stretch, which shall have about the same or higher potency (as defined herein);
e) SEQ ID NO: 121, and
f) an amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 121, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (such as measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference an amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Select from stretches of amino acid residues that should have about the same or higher potency (as defined herein). Comprises or consists essentially of at least two amino acid sequences and / or Nanobodies®, wherein the stretch of amino acid residues corresponding to one of a) and b) is always an amino acid according to the invention. Embodiments E-7 to E wherein the second amino acid residue stretch is as present in the sequence and the second amino acid residue stretch is as selected from one of c), d), e) and f). The polyvalent polypeptide according to any one of -10.
Aspect E-12:
a) SEQ ID NO: 98,
b) an amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 98, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (as said affinity is measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference the amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Select from stretches of amino acid residues that should have about the same or higher potency (as defined herein) Is, at least one amino acid residue stretch,
c) SEQ ID NO: 102,
d) a stretch of amino acid residues having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 102,
i) the amino acid residue stretch has aspartic acid (Asp, D) at position 6 (position 54 determined by Kabat numbering),
ii) the amino acid sequence comprising the amino acid residue stretch has the same, substantially the same or higher affinity as compared to the amino acid sequence comprising the amino acid residue stretch having no three, two or one amino acid difference ( The affinity is as measured by surface plasmon resonance) and binds to protein F of hRSV and / or the amino acid sequence comprising said stretch of amino acid residues differs by three, two or one amino acid Selected from the group consisting of amino acid residue stretches that have the same, approximately the same, or higher potency (as defined herein) as compared to an amino acid sequence comprising the above amino acid residue stretches without A second stretch of amino acid residues,
e) SEQ ID NO: 121,
f) an amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 121, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (such as measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference an amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Stretches of amino acid residues which should be of about the same or higher potency (as defined herein). Any of embodiments E-7 to E-11, comprising or consisting essentially of at least two amino acid sequences and / or Nanobodies®, including a third amino acid residue stretch selected from the group A multivalent polypeptide according to any one of the above.
Aspect E-13: Any one of Aspects E-7 to E-12, comprising or consisting essentially of at least two amino acid sequences and / or Nanobodies®, including at least SEQ ID NO: 102. A multivalent polypeptide according to any one of the preceding claims.
Aspect E-14: at least SEQ ID NO: 102;
a) SEQ ID NO: 98,
b) an amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 98, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (as said affinity is measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference the amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; An amino acid residue stretch, which shall have about the same or higher potency (as defined herein);
c) SEQ ID NO: 121, and
d) An amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 121, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (as said affinity is measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference the amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Select from stretches of amino acid residues that should have about the same or higher potency (as defined herein). Embodiments E-7 to E-13 comprising or consisting essentially of at least two amino acid sequences and / or Nanobodies®, including at least one stretch of amino acid residues (CDR sequences). A multivalent polypeptide according to any one of the above.
Aspect E-15: At least SEQ ID NO: 102;
a) SEQ ID NO: 98,
b) an amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 98, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (as said affinity is measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference the amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Select from stretches of amino acid residues that should have about the same or higher potency (as defined herein) Is a CDR1 sequence selected from at least one amino acid residue stretch,
c) SEQ ID NO: 121, and
d) An amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 121, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (as said affinity is measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference the amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Select from stretches of amino acid residues that should have about the same or higher potency (as defined herein). A multivalent according to any one of aspects E-7 to E-14, comprising or consisting essentially of at least two amino acid sequences and / or Nanobodies®, comprising the CDR3 sequence to be made. Polypeptide.
Aspect E-16: At least two amino acid sequences and / or Nanobodies comprising at least SEQ ID NO: 102 and at least one amino acid residue stretch (CDR sequence) selected from SEQ ID NO: 98 and SEQ ID NO: 121 The multivalent polypeptide according to any one of aspects E-7 to E-15, comprising or consisting essentially of).
Aspect E-17: Aspects E-7-E comprising or consisting essentially of at least two amino acid sequences and / or Nanobodies®, including SEQ ID NO: 98, SEQ ID NO: 102, and SEQ ID NO: 121. The multivalent polypeptide according to any one of -16.
Aspect E-18: Polyvalent polypeptide according to any one of the aspects E-8 to E-17, wherein said at least two amino acid sequences and / or Nanobodies® are identical.
Aspect E-19:
a) SEQ ID NO: 60 to SEQ ID NO: 76
b) an amino acid sequence having only one amino acid difference from one of SEQ ID NO: 60 to SEQ ID NO: 76, preferably only two, more preferably only one;
i) the amino acid sequence has glutamine (Gln, Q) at position 105 (the position is determined by Kabat numbering);
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
A multivalent polypeptide according to any one of aspects E-7 to E-9, comprising or consisting essentially of at least two amino acid sequences and / or Nanobodies® selected from the group consisting of:
Aspect E-20: Polyvalent polypeptide according to aspect E-19, wherein said at least two amino acid sequences and / or Nanobodies® are identical.
Aspect E-21: Aspect E, comprising or consisting essentially of at least two identical amino acid sequences and / or Nanobodies® selected from one of SEQ ID NO: 60-76. The polyvalent polypeptide according to -20.
Aspect E-22:
a) SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76
b) only three, preferably only two and more preferably only one amino acid difference from one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75 and SEQ ID NO: 76 An amino acid sequence having
i) the amino acid sequence has glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and / or arginine (Arg, R) at position 83 (the above positions are determined by Kabat numbering; Desired),
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
A multivalent polypeptide according to any one of aspects E-7 to E-9, comprising or consisting essentially of at least two amino acid sequences and / or Nanobodies® selected from the group consisting of:
Aspect E-23:
a) SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76
b) only three, preferably only two and more preferably only one amino acid difference from one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75 and SEQ ID NO: 76 An amino acid sequence having
i) the amino acid sequence has glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and arginine (Arg, R) at position 83 (the above positions are determined by Kabat numbering) ),
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
A multivalent polypeptide according to aspect E-22, comprising or consisting essentially of at least two amino acid sequences and / or Nanobodies® selected from the group consisting of:
Aspect E-24: A multivalent polypeptide according to aspect E-22 or E-23, wherein said at least two amino acid sequences and / or Nanobodies® are identical.
Aspect E-25: At least two identical amino acid sequences and / or Nanobodies selected from one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76 A multivalent polypeptide according to aspect E-24, which comprises or consists essentially of RTM.
Aspect E-26:
a) SEQ ID NO: 65 and SEQ ID NO: 76,
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NO: 65 and SEQ ID NO: 76,
i) the amino acid sequence is aspartic acid (Asp, D) at position 54, glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and / or arginine (Arg, R) at position 83; (The position is determined by Kabat numbering),
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
A multivalent polypeptide according to any one of aspects E-7 to E-9, comprising or consisting essentially of at least two amino acid sequences and / or Nanobodies® selected from the group consisting of:
Aspect E-27:
a) SEQ ID NO: 65 and SEQ ID NO: 76,
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NO: 65 and SEQ ID NO: 76,
i) the amino acid sequence has aspartic acid (Asp, D) at position 54, glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and arginine (Arg, R) at position 83; (The above position is obtained by Kabat numbering)
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
The multivalent polypeptide according to aspect E-26, comprising or consisting essentially of at least two amino acid sequences and / or Nanobodies® selected from the group consisting of:
Aspect E-28: A multivalent polypeptide according to aspect E-26 or E-27, wherein the at least two amino acid sequences and / or Nanobodies® are identical.
Aspect E-29: Aspect according to aspect E-28, comprising or consisting essentially of at least two identical amino acid sequences and / or Nanobodies® selected from SEQ ID NO: 65 and SEQ ID NO: 76. Multivalent polypeptide.
Aspect E-30:
a) SEQ ID NO: 146 to SEQ ID NO: 153
b) an amino acid sequence having only one, preferably two, more preferably only one amino acid difference from one of SEQ ID NOs: 146 to 153,
i) the amino acid sequence has proline (Pro, P) at position 14, arginine (Arg, R) at position 19, leucine (Leu, L) at position 20, and leucine (Leu, L) at position 108 (The above position is determined by Kabat numbering),
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
A multivalent polypeptide according to any one of aspects E-7 to E-9, comprising or consisting essentially of at least two amino acid sequences and / or Nanobodies® selected from the group consisting of:
Aspect E-31: Multivalent polypeptide according to aspect E-30, wherein said at least two amino acid sequences and / or Nanobodies® are identical.
Aspect E-32: Aspect E comprising or consisting essentially of at least two identical amino acid sequences and / or Nanobodies® selected from one of SEQ ID NOs: 146-153. The multivalent polypeptide according to -31.
Aspect E-33:
a) SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153,
i) the amino acid sequence comprises proline (Pro, P) at position 14, arginine (Arg, R) at position 19, leucine (Leu, L) at position 20, and leucine (Leu, L) at position 108; Arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85, and / or glutamine (Gln, Q) at position 105 (the position is determined by Kabat numbering);
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
A multivalent polypeptide according to any one of aspects E-7 to E-9, comprising or consisting essentially of at least two amino acid sequences and / or Nanobodies® selected from the group consisting of:
Aspect E-34:
a) SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153,
i) the amino acid sequence is
SEQ ID NO: 146 (this amino acid sequence preferably has glutamine (Gln, Q) at position 105);
SEQ ID NO: 147 (this amino acid sequence preferably has arginine (Arg, R) at position 83 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 148 (this amino acid sequence preferably has glutamic acid (Glu, E) at position 85 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 149 (this amino acid sequence preferably has arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 151 (this amino acid sequence preferably has arginine (Arg, R) at position 83);
SEQ ID NO: 152 (this amino acid sequence preferably has glutamic acid (Glu, E) at position 85);
SEQ ID NO: 153 (this amino acid sequence preferably has arginine (Arg, R) at position 83 and glutamic acid (Glu, E) at position 85)
The amino acid sequence has a proline (Pro, P) at position 14 and an arginine (Arg, R) at position 19, as in the case of having only 3, preferably 2, and more preferably only 1 amino acid difference. Leucine (Leu, L) at position 20, Leucine (Leu, L) at position 108, arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85, and / or glutamine at position 105 (Gln, Q) (the position is determined by Kabat numbering),
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
A multivalent polypeptide according to aspect E-33, comprising or consisting essentially of at least two amino acid sequences and / or Nanobodies® selected from the group consisting of:
Aspect E-35: Polyvalent polypeptide according to aspect E-33 or E-34, wherein the at least two amino acid sequences and / or Nanobodies® are identical.
Aspect E-36: At least two identical members selected from SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 147, SEQ ID NO: 149, and SEQ ID NO: 153 The multivalent polypeptide according to aspect E-35, comprising or consisting essentially of the amino acid sequence of and / or Nanobody®.
Aspect E-37: One or more (eg, 2, 3, 4, 5, 6, 7, 8, or 9, 10, 11, or 12) amino acid residues A sequence selected from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and at least two amino acids with a mutation selected from SEQ ID NO: 5 and / or a body with at least two amino acids selected from Gly54Asp. A multivalent polypeptide comprising or consisting essentially of (registered trademark).
Aspect E-38: Amino acid residue is mutated with at least 5 sequences having Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54D. The multivalent polypeptide according to aspect E-37, which comprises or consists essentially of an amino acid sequence and / or Nanobody®.
Aspect E-39: wherein one or more (eg, 2, 3, 4, 5, 6, 7, or 8) amino acid residues are Ser19R, Ile20Leu, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Aspect E-37, comprising or consisting essentially of at least two amino acid sequences having SEQ ID NO: 5 and / or Nanobody®, mutated selected from Arg105Gln and Gly54D. Multivalent polypeptide.
Aspect E-40: Sequence wherein one or more (eg, 2, 3, 4, or 5) amino acid residues are mutated selected from Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54D. The multivalent polypeptide according to aspect E-37, comprising or consisting essentially of at least two amino acid sequences having the number 5, and / or Nanobody®.
Aspect E-41: The amino acid residue comprises at least two amino acid sequences having SEQ ID NO: 5 and / or Nanobody®, mutated Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54D; Or consisting essentially of the multivalent polypeptide of embodiment E-40.
Aspect E-42: wherein one or more (eg, two, three or four) amino acid residues has a SEQ ID NO: 5, mutated selected from Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu. A multivalent polypeptide comprising or consisting essentially of at least two amino acid sequences and / or Nanobodies®.
Embodiment E-43: wherein one or more (eg, 2, 3, 4, 5, 6, or 7) amino acid residues are from Ala14Pro, Ser19Arg, Ile20Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu. A multivalent polypeptide comprising or consisting essentially of at least two amino acid sequences having SEQ ID NO: 5 and / or Nanobody®, having a selected mutation.
Aspect E-44:
The amino acid residue is
Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Gly78Leu, Ala83Arg, Asp85Glu, and Arg105Gln,
Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54Asp,
Gly54Asp,
Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Ala83Arg,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Asp85Glu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Arg105Gln,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Asp85Glu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Arg105Gln,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Asp85Glu, and Arg105Gln, or
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, Asp85Glu, and Arg105Gln
A multivalent polypeptide according to aspect E-37, comprising or consisting essentially of at least two amino acid sequences having SEQ ID NO: 5 and / or Nanobody®, mutated to:
Aspect E-45: At least three amino acid sequences according to any one of Aspects A-1 to A-29, Aspects C-1 to C-38, and Aspects W-1 to W-38, and / or Includes or consists essentially of Nanobody (R) according to any one of Aspects B-1 to B-18, Aspects D-1 to D-38, and Aspects X-1 to X-38. The multivalent polypeptide according to aspect E-7.
Aspect E-46: A multivalent polypeptide according to aspect E-45, wherein the at least three amino acid sequences and / or Nanobodies® are identical.
Aspect E-47:
a) SEQ ID NO: 102,
b) a stretch of amino acid residues having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 102,
i) the amino acid residue stretch has aspartic acid (Asp, D) at position 6 (position 54 determined by Kabat numbering),
ii) the amino acid sequence comprising the amino acid residue stretch has the same, substantially the same or higher affinity as compared to the amino acid sequence comprising the amino acid residue stretch having no three, two or one amino acid difference ( The affinity is as measured by surface plasmon resonance) and binds to protein F of hRSV and / or the amino acid sequence comprising said stretch of amino acid residues differs by three, two or one amino acid An amino acid selected from an amino acid residue stretch that has the same, approximately the same, or a higher potency (as defined herein) as compared to an amino acid sequence comprising the above amino acid residue stretch without Whether it comprises at least three amino acid sequences and / or Nanobodies®, including at least one residue stretch Or which consists essentially of, multivalent polypeptide according to embodiment E-45 or E-46.
Aspect E-48:
a) SEQ ID NO: 102,
b) a stretch of amino acid residues having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 102,
i) the amino acid residue stretch has aspartic acid (Asp, D) at position 6 (position 54 determined by Kabat numbering),
ii) the amino acid sequence comprising the amino acid residue stretch has the same, substantially the same or higher affinity as compared to the amino acid sequence comprising the amino acid residue stretch having no three, two or one amino acid difference ( The affinity is as measured by surface plasmon resonance) and binds to protein F of hRSV and / or the amino acid sequence comprising said stretch of amino acid residues differs by three, two or one amino acid Selected from amino acid residue stretches that have the same, approximately the same, or higher potency (as defined herein) as compared to an amino acid sequence comprising the amino acid residue stretch without At least one amino acid residue stretch,
At least one stretch,
c) SEQ ID NO: 98;
d) An amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 98, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (as said affinity is measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference the amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; An amino acid residue stretch, which shall have about the same or higher potency (as defined herein);
e) SEQ ID NO: 121, and
f) an amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 121, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (such as measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference an amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Select from stretches of amino acid residues that should have about the same or higher potency (as defined herein). Comprises or consists essentially of at least three amino acid sequences and / or Nanobodies®, wherein the stretch of amino acid residues corresponding to one of a) and b) is always an amino acid according to the invention. Aspect E-45 or E, wherein the second stretch of amino acid residues is as present in the sequence, and wherein the second amino acid residue stretch is as selected from one of c), d), e) and f). A multivalent polypeptide according to -46.
Aspect E-49:
a) SEQ ID NO: 98, and
b) an amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 98, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (as said affinity is measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference the amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Select from stretches of amino acid residues that should have about the same or higher potency (as defined herein) Is, at least one amino acid residue stretch,
c) SEQ ID NO: 102, and
d) a stretch of amino acid residues having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 102,
i) the amino acid residue stretch has aspartic acid (Asp, D) at position 6 (position 54 determined by Kabat numbering),
ii) the amino acid sequence comprising the amino acid residue stretch has the same, substantially the same or higher affinity as compared to the amino acid sequence comprising the amino acid residue stretch having no three, two or one amino acid difference ( The affinity is as measured by surface plasmon resonance) and binds to protein F of hRSV and / or the amino acid sequence comprising said stretch of amino acid residues differs by three, two or one amino acid Selected from the group consisting of amino acid residue stretches that have the same, approximately the same, or higher potency (as defined herein) as compared to an amino acid sequence comprising the above amino acid residue stretches without A second stretch of amino acid residues,
e) SEQ ID NO: 121, and
f) an amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 121, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (such as measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference an amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Stretches of amino acid residues which should be of about the same or higher potency (as defined herein). Aspect E-45 or E-46, comprising or consisting essentially of at least three amino acid sequences and / or Nanobodies®, including a third amino acid residue stretch selected from the group Multivalent polypeptide.
Aspect E-50: Multivalent poly according to aspect E-45 or E-46, comprising or consisting essentially of at least three amino acid sequences comprising at least SEQ ID NO: 102 and / or Nanobody®. peptide.
Aspect E-51: at least SEQ ID NO: 102;
a) SEQ ID NO: 98,
b) an amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 98, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (as said affinity is measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference the amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; An amino acid residue stretch, which shall have about the same or higher potency (as defined herein);
c) SEQ ID NO: 121, and
d) An amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 121, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (as said affinity is measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference the amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Select from stretches of amino acid residues that should have about the same or higher potency (as defined herein). Embodiment E-45 or E-46 comprising or consisting essentially of at least three amino acid sequences and / or Nanobodies®, including at least one stretch of amino acid residues (CDR sequences). 2. The multivalent polypeptide according to item 1.
Aspect E-52: at least SEQ ID NO: 102;
a) SEQ ID NO: 98,
b) an amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 98, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (as said affinity is measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference the amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Select from stretches of amino acid residues that should have about the same or higher potency (as defined herein) Is a CDR1 sequence selected from at least one amino acid residue stretch,
c) SEQ ID NO: 121, and
d) An amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 121, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (as said affinity is measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference the amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Select from stretches of amino acid residues that should have about the same or higher potency (as defined herein). A CDR3 sequence that is, it contains at least three amino acid sequences and / or Nanobody (TM), consists essentially or therefrom, multivalent polypeptide according to embodiment E-45 or E-46.
Aspect E-53: at least three amino acid sequences and / or Nanobodies comprising at least SEQ ID NO: 102 and at least one amino acid residue stretch (CDR sequence) selected from SEQ ID NO: 98 and SEQ ID NO: 121 A multivalent polypeptide according to aspect E-45 or E-46, comprising or consisting essentially of).
Aspect E-54: Aspects E-45 or E comprising or consisting essentially of at least three amino acid sequences and / or Nanobodies®, including SEQ ID NO: 98, SEQ ID NO: 102, and SEQ ID NO: 121. A multivalent polypeptide according to -46.
Aspect E-55: Polyvalent polypeptide according to any one of the aspects E-47 to E-53, wherein the at least three amino acid sequences and / or Nanobodies® are identical.
Aspect E-56:
a) SEQ ID NO: 60 to SEQ ID NO: 76
b) an amino acid sequence having only one amino acid difference from one of SEQ ID NO: 60 to SEQ ID NO: 76, preferably only two, more preferably only one;
i) the amino acid sequence has glutamine (Gln, Q) at position 105 (the position is determined by Kabat numbering);
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
A multivalent polypeptide according to aspect E-45 or E-46, comprising or consisting essentially of at least three amino acid sequences and / or Nanobodies® selected from the group consisting of:
Aspect E-57: Polyvalent polypeptide according to aspect E-56, wherein the at least three amino acid sequences and / or Nanobodies® are identical.
Aspect E-58: Aspect E comprising, or consisting essentially of, at least three identical amino acid sequences and / or Nanobodies® selected from one of SEQ ID NO: 60-SEQ ID NO: 76. The multivalent polypeptide according to -57.
Aspect E-59:
a) SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76
b) only three, preferably only two and more preferably only one amino acid difference from one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75 and SEQ ID NO: 76 I) the amino acid sequence has glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and arginine (Arg, R) at position 83 (as described above); Is determined by Kabat numbering),
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
A multivalent polypeptide according to aspect E-45 or E-46, comprising or consisting essentially of at least three amino acid sequences and / or Nanobodies® selected from the group consisting of:
Aspect E-60:
a) SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76
b) only three, preferably only two and more preferably only one amino acid difference from one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75 and SEQ ID NO: 76 An amino acid sequence having
i) the amino acid sequence has glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and arginine (Arg, R) at position 83 (the above positions are determined by Kabat numbering) ),
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
The multivalent polypeptide according to aspect E-59, comprising or consisting essentially of at least three amino acid sequences and / or Nanobodies® selected from the group consisting of:
Aspect E-61: A multivalent polypeptide according to aspect E-59 or E-60, wherein said at least three amino acid sequences and / or Nanobodies® are identical.
Aspect E-62: at least three identical amino acid sequences and / or Nanobodies selected from one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76 A multivalent polypeptide according to aspect E-61, which comprises or consists essentially of (registered trademark).
Aspect E-63:
a) SEQ ID NO: 65 and SEQ ID NO: 76,
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NO: 65 and SEQ ID NO: 76,
i) the amino acid sequence is aspartic acid (Asp, D) at position 54, glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and / or arginine (Arg, R) at position 83; (The position is determined by Kabat numbering),
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
A multivalent polypeptide according to aspect E-45 or E-46, comprising or consisting essentially of at least three amino acid sequences and / or Nanobodies® selected from the group consisting of:
Aspect E-64:
a) SEQ ID NO: 65 and SEQ ID NO: 76,
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NO: 65 and SEQ ID NO: 76,
i) the amino acid sequence has aspartic acid (Asp, D) at position 54, glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and arginine (Arg, R) at position 83; (The above position is obtained by Kabat numbering)
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
The multivalent polypeptide according to aspect E-63, comprising or consisting essentially of at least three amino acid sequences and / or Nanobodies® selected from the group consisting of:
Aspect E-65: A multivalent polypeptide according to aspect E-63 or E-64, wherein the at least three amino acid sequences and / or Nanobodies® are identical.
Aspect E-66: Aspect according to aspect E-65, comprising or consisting essentially of at least three identical amino acid sequences and / or Nanobodies® selected from SEQ ID NO: 65 and SEQ ID NO: 76. Multivalent polypeptide.
Aspect E-67:
a) SEQ ID NO: 146 to SEQ ID NO: 153
b) an amino acid sequence having only one, preferably two, more preferably only one amino acid difference from one of SEQ ID NOs: 146 to 153,
i) the amino acid sequence has proline (Pro, P) at position 14, arginine (Arg, R) at position 19, leucine (Leu, L) at position 20, and leucine (Leu, L) at position 108 (The above position is determined by Kabat numbering),
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
A multivalent polypeptide according to aspect E-45 or E-46, comprising or consisting essentially of at least three amino acid sequences and / or Nanobodies® selected from the group consisting of:
Aspect E-68: Polyvalent polypeptide according to aspect E-67, wherein the at least three amino acid sequences and / or Nanobodies® are identical.
Aspect E-69: Aspect E comprising, or consisting essentially of, at least three identical amino acid sequences and / or Nanobodies® selected from one of SEQ ID NOs: 146-153. The multivalent polypeptide according to -68.
Aspect E-70:
a) SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153,
i) the amino acid sequence comprises proline (Pro, P) at position 14, arginine (Arg, R) at position 19, leucine (Leu, L) at position 20, and leucine (Leu, L) at position 108; Arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85, and / or glutamine (Gln, Q) at position 105 (the position is determined by Kabat numbering);
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
A multivalent polypeptide according to aspect E-45 or E-46, comprising or consisting essentially of at least three amino acid sequences and / or Nanobodies® selected from the group consisting of:
Aspect E-71:
a) SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153,
i) the amino acid sequence is
SEQ ID NO: 146 (this amino acid sequence preferably has glutamine (Gln, Q) at position 105);
SEQ ID NO: 147 (this amino acid sequence preferably has arginine (Arg, R) at position 83 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 148 (this amino acid sequence preferably has glutamic acid (Glu, E) at position 85 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 149 (this amino acid sequence preferably has arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 151 (this amino acid sequence preferably has arginine (Arg, R) at position 83);
SEQ ID NO: 152 (this amino acid sequence preferably has glutamic acid (Glu, E) at position 85);
SEQ ID NO: 153 (this amino acid sequence preferably has arginine (Arg, R) at position 83 and glutamic acid (Glu, E) at position 85)
The amino acid sequence has a proline (Pro, P) at position 14 and an arginine (Arg, R) at position 19, as in the case of having only 3, preferably 2, and more preferably only 1 amino acid difference. Leucine (Leu, L) at position 20, Leucine (Leu, L) at position 108, arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85, and / or glutamine at position 105 (Gln, Q) (the position is determined by Kabat numbering),
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
A multivalent polypeptide according to aspect E-70, comprising or consisting essentially of at least three amino acid sequences and / or Nanobodies® selected from the group consisting of:
Aspect E-72: A multivalent polypeptide according to aspect E-70 or E-71, wherein said at least three amino acid sequences and / or Nanobodies® are identical.
Aspect E-73: at least three identical members selected from SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 147, SEQ ID NO: 149, and SEQ ID NO: 153 A multivalent polypeptide according to aspect E-72, which comprises or consists essentially of the amino acid sequence of and / or Nanobody®.
Aspect E-74: one or more (eg, 2, 3, 4, 5, 6, 7, 8, or 9, 10, 11, or 12) amino acid residues A sequence selected from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu and / or Gly54Asp having a mutation selected from the group consisting of SEQ ID NO: 5 and / or having at least three amino acids. A multivalent polypeptide comprising or consisting essentially of (registered trademark).
Aspect E-75: Amino acid residue has at least 5 mutations of at least 5 sequences having Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly108D. The multivalent polypeptide according to aspect E-74, comprising or consisting essentially of an amino acid sequence and / or Nanobody®.
Aspect E-76: wherein one or more (eg, 2, 3, 4, 5, 6, 7, or 8) amino acid residues are Ser19R, Ile20Leu, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Aspect E-74, comprising or consisting essentially of at least three amino acid sequences having SEQ ID NO: 5 and / or Nanobody®, mutated selected from Arg105Gln, and Gly54D. Multivalent polypeptide.
Aspect E-77: A sequence wherein one or more (eg, 2, 3, 4, or 5) amino acid residues are mutated selected from Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54D. The multivalent polypeptide according to aspect E-74, comprising or consisting essentially of at least three amino acid sequences having the number 5, and / or Nanobody®.
Aspect E-78: wherein the amino acid residue comprises at least three amino acid sequences and / or Nanobodies® having SEQ ID NO: 5, mutated Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54D, Or a multivalent polypeptide according to aspect E-77 consisting essentially of, or consisting of,
Aspect E-79: having one or more (e.g., two, three or four) amino acid residues having a SEQ ID NO: 5, mutated selected from Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu. A multivalent polypeptide comprising or consisting essentially of at least three amino acid sequences and / or Nanobodies®.
Aspect E-80: One or more (eg, 2, 3, 4, 5, 6, or 7) amino acid residues are from Ala14Pro, Ser19Arg, Ile20Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu. A multivalent polypeptide comprising or consisting essentially of at least three amino acid sequences having SEQ ID NO: 5 and / or Nanobody®, having a selected mutation.
Aspect E-81:
The amino acid residue is
Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Gly78Leu, Ala83Arg, Asp85Glu, and Arg105Gln,
Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54Asp,
Gly54Asp,
Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Ala83Arg,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Asp85Glu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Arg105Gln,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Asp85Glu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Arg105Gln,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Asp85Glu, and Arg105Gln, or
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, Asp85Glu, and Arg105Gln
The multivalent polypeptide according to aspect E-74, which comprises or consists essentially of at least three amino acid sequences having SEQ ID NO: 5 and / or Nanobody®, mutated to:
Aspect E-82:
a) SEQ ID NO: 138 to SEQ ID NO: 141 and SEQ ID NO: 154 to SEQ ID NO: 157;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NO: 138 to SEQ ID NO: 141,
i) the amino acid sequence has aspartic acid (Asp, D) at position 1 (the position is determined by Kabat numbering);
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
A multivalent polypeptide according to any one of aspects E-7 to E-9, comprising or consisting essentially of at least one amino acid sequence and / or Nanobody (R) selected from the group consisting of:
Aspect E-83: Comprising or comprising at least one amino acid sequence and / or Nanobody® selected from one of SEQ ID NOs: 138-141 and SEQ ID NOs: 154-157. A multivalent polypeptide according to aspect E-82, wherein the polypeptide is
Embodiment E-84: A polyvalent polyprotein comprising or consisting essentially of at least one amino acid sequence having SEQ ID NO: 5 and / or Nanobody®, wherein glutamic acid at position 1 has been changed to aspartic acid. peptide.
Aspect E-85 Multivalent poly comprising or consisting essentially of at least one amino acid sequence having SEQ ID NO: 62 and / or Nanobody®, wherein glutamic acid at position 1 has been changed to aspartic acid peptide.
Aspect E-86: Multivalent poly comprising or consisting essentially of at least one amino acid sequence having SEQ ID NO: 65 and / or Nanobody®, wherein glutamic acid at position 1 has been changed to aspartic acid peptide.
Aspect E-87: Multivalent poly comprising or consisting essentially of at least one amino acid sequence having SEQ ID NO: 76 and / or Nanobody®, wherein glutamic acid at position 1 has been changed to aspartic acid. peptide.
Embodiment E-88: Multivalent poly comprising or consisting essentially of at least one amino acid sequence having SEQ ID NO: 75 and / or Nanobody®, wherein glutamic acid at position 1 has been changed to aspartic acid peptide.
Aspect E-89: Polyvalent polyvalent polymer comprising or consisting essentially of at least one amino acid sequence having SEQ ID NO: 147 and / or Nanobody®, wherein glutamic acid at position 1 has been changed to aspartic acid peptide.
Aspect E-90. Multivalent poly comprising or consisting essentially of at least one amino acid sequence and / or Nanobody® having SEQ ID NO: 149, wherein glutamic acid at position 1 has been changed to aspartic acid. peptide.
Aspect E-91 Multivalent poly comprising or consisting essentially of at least one amino acid sequence and / or Nanobody® having SEQ ID NO: 153, wherein glutamic acid at position 1 has been changed to aspartic acid peptide.
Aspect E-92: one or more (e.g., 2, 3, 4, 5, 6, 7, 8, or 9, 10, 11, or 12) amino acid residues , Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and a mutation in which the amino acid at the amino acid position Glu54Asp has a mutation selected from the following: A multivalent polypeptide comprising, or consisting essentially of, at least one amino acid sequence having the number 5, and / or Nanobody®.
Aspect E-93: Amino acid residue has mutation at Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly108D, and amino acid residue is glutamic acid at amino acid position, and amino acid residue is glutamic acid at amino acid position. The multivalent polypeptide according to aspect E-92, which comprises, or consists essentially of, at least one amino acid sequence and / or Nanobody® having an altered SEQ ID NO: 5.
Embodiment E-94: wherein one or more (eg, 2, 3, 4, 5, 6, 7, or 8) amino acid residues are Ser19R, Ile20Leu, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Contains at least one amino acid sequence having SEQ ID NO: 5 and / or Nanobody®, which has a mutation selected from Arg105Gln, and Gly54D, wherein glutamic acid at position 1 has been changed to aspartic acid. Or a multivalent polypeptide according to aspect E-92, consisting essentially of, or consisting of,
Aspect E-95: one or more (eg, 2, 3, 4, or 5) amino acid residues have a mutation selected from Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54D; Aspect E-92, comprising or consisting essentially of at least one amino acid sequence having SEQ ID NO: 5 and / or Nanobody®, wherein glutamic acid at position 1 has been changed to aspartic acid. Multivalent polypeptide.
Aspect E-96: At least one having SEQ ID NO: 5, wherein the amino acid residue has been mutated for Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54D, and wherein glutamic acid at position 1 has been changed to aspartic acid. The multivalent polypeptide according to aspect E-95, which comprises or consists essentially of the amino acid sequence and / or Nanobody®.
Aspect E-97: One or more (eg, two, three or four) amino acid residues have been mutated selected from Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu, and glutamic acid at position 1 has A multivalent polypeptide comprising or consisting essentially of at least one amino acid sequence having SEQ ID NO: 5 and / or Nanobody®, which has been changed to aspartic acid.
Aspect E-98: One or more (eg, 2, 3, 4, 5, 6, or 7) amino acid residues are derived from Ala14Pro, Ser19Arg, Ile20Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu. At least one amino acid sequence having SEQ ID NO: 5 and / or comprising or consisting essentially of a mutated glutamic acid having been changed to aspartic acid, which has undergone a selected mutation. Polyvalent polypeptide.
Aspect E-99:
The amino acid residue is
Glu1Asp,
Glu1Asp, Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Glu1Asp, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Glu1Asp, Gly78Leu, Ala83Arg, Asp85Glu, and Arg105Gln;
Glu1Asp, Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Glu1Asp, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Glu1Asp, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54Asp,
Glu1Asp and Gly54Asp,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Ala83Arg;
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Asp85Glu,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Arg105Gln,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Asp85Glu,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Arg105Gln,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Asp85Glu, and Arg105Gln, or
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, Asp85Glu, and Arg105Gln
The multivalent polypeptide according to aspect E-92, comprising or consisting essentially of at least one amino acid sequence having SEQ ID NO: 5 and / or Nanobody®, mutated to:
Aspect E-100: Two amino acid sequences according to any one of Aspects A-1 to A-29, Aspects C-1 to C-38, and Aspects W-1 to W-38, and / or Aspects Comprising or consisting essentially of Nanobody (R) according to any one of B-1 to B-18, Aspects D-1 to D-38, and Aspects X-1 to X-38, The bivalent polypeptide according to aspect E-7.
Aspect E-101: The bivalent polypeptide of Aspect E-100, wherein the two amino acid sequences and / or Nanobodies® are identical.
Aspect E-102:
a) SEQ ID NO: 102,
b) a stretch of amino acid residues having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 102,
i) the amino acid residue stretch has aspartic acid (Asp, D) at position 6 (position 54 determined by Kabat numbering),
ii) the amino acid sequence comprising the amino acid residue stretch has the same, substantially the same or higher affinity as compared to the amino acid sequence comprising the amino acid residue stretch having no three, two or one amino acid difference ( The affinity is as measured by surface plasmon resonance) and binds to protein F of hRSV and / or the amino acid sequence comprising said stretch of amino acid residues differs by three, two or one amino acid An amino acid selected from an amino acid residue stretch that has the same, approximately the same, or a higher potency (as defined herein) as compared to an amino acid sequence comprising the above amino acid residue stretch without Comprising or comprising two amino acid sequences comprising at least one residue stretch and / or Nanobody® Luo consisting essentially, bivalent polypeptide according to aspect E-100 or E-101.
Aspect E-103:
a) SEQ ID NO: 102,
b) a stretch of amino acid residues having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 102,
i) the amino acid residue stretch has aspartic acid (Asp, D) at position 6 (position 54 determined by Kabat numbering),
ii) the amino acid sequence comprising the amino acid residue stretch has the same, substantially the same or higher affinity as compared to the amino acid sequence comprising the amino acid residue stretch having no three, two or one amino acid difference ( The affinity is as measured by surface plasmon resonance) and binds to protein F of hRSV and / or the amino acid sequence comprising said stretch of amino acid residues differs by three, two or one amino acid Selected from amino acid residue stretches that have the same, approximately the same, or higher potency (as defined herein) as compared to an amino acid sequence comprising the amino acid residue stretch without At least one amino acid residue stretch,
At least one stretch,
c) SEQ ID NO: 98;
d) An amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 98, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (as said affinity is measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference the amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; An amino acid residue stretch, which shall have about the same or higher potency (as defined herein);
e) SEQ ID NO: 121, and
f) an amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 121, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (such as measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference an amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Select from stretches of amino acid residues that should have about the same or higher potency (as defined herein). The amino acid sequence comprising or consisting essentially of two amino acid sequences and / or Nanobodies®, wherein the amino acid residue stretch corresponding to one of a) and b) is always an amino acid sequence according to the invention. E-100 or E-, wherein the second amino acid residue stretch is as selected from one of c), d), e) and f). 101. The bivalent polypeptide according to 101.
Aspect E-104:
a) SEQ ID NO: 98,
b) an amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 98, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (as said affinity is measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference the amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Select from stretches of amino acid residues that should have about the same or higher potency (as defined herein) Is, at least one amino acid residue stretch,
c) SEQ ID NO: 102,
d) a stretch of amino acid residues having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 102,
i) the amino acid residue stretch has aspartic acid (Asp, D) at position 6 (position 54 determined by Kabat numbering),
ii) the amino acid sequence comprising the amino acid residue stretch has the same, substantially the same or higher affinity as compared to the amino acid sequence comprising the amino acid residue stretch having no three, two or one amino acid difference ( The affinity is as measured by surface plasmon resonance) and binds to protein F of hRSV and / or the amino acid sequence comprising said stretch of amino acid residues differs by three, two or one amino acid Selected from the group consisting of amino acid residue stretches that have the same, approximately the same, or higher potency (as defined herein) as compared to an amino acid sequence comprising the above amino acid residue stretches without A second stretch of amino acid residues,
e) SEQ ID NO: 121, and
f) an amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 121, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (such as measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference an amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Stretches of amino acid residues which should be of about the same or higher potency (as defined herein). Aspect E-100 or E-101, comprising or consisting essentially of two amino acid sequences and / or Nanobodies®, including a third amino acid residue stretch selected from the group Bivalent polypeptide.
Aspect E-105: Bivalent polypeptide according to aspect E-100 or E-101, comprising or consisting essentially of two amino acid sequences comprising SEQ ID NO: 102 and / or Nanobody®. .
Aspect E-106: at least SEQ ID NO: 102;
a) SEQ ID NO: 98,
b) an amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 98, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (as said affinity is measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference the amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; An amino acid residue stretch, which shall have about the same or higher potency (as defined herein);
c) SEQ ID NO: 121, and
d) An amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 121, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (as said affinity is measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference the amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Select from stretches of amino acid residues that should have about the same or higher potency (as defined herein). Aspect E-100 or E-101 comprising, or consisting essentially of, two amino acid sequences and / or Nanobodies®, comprising at least one stretch of amino acid residues (CDR sequences). A bivalent polypeptide as described.
Aspect E-107: at least SEQ ID NO: 102;
a) SEQ ID NO: 98, and
b) an amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 98, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (as said affinity is measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference the amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Select from stretches of amino acid residues that should have about the same or higher potency (as defined herein) Is a CDR1 sequence selected from at least one amino acid residue stretch,
c) SEQ ID NO: 121, and
d) An amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 121, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (as said affinity is measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference the amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Select from stretches of amino acid residues that should have about the same or higher potency (as defined herein). A CDR3 sequence that is, it contains two amino acid sequences and / or Nanobody (TM), consists essentially or therefrom, bivalent polypeptide according to aspect E-100 or E-101.
Aspect E-108: two amino acid sequence and / or Nanobody® comprising at least SEQ ID NO: 102 and at least one amino acid residue stretch (CDR sequence) selected from SEQ ID NO: 98 and SEQ ID NO: 121 A bivalent polypeptide according to aspect E-100 or E-101, comprising or consisting essentially of
Aspect E-109: Aspect E-100 or E-, comprising or consisting essentially of two amino acid sequences and / or Nanobodies®, including SEQ ID NO: 98, SEQ ID NO: 102, and SEQ ID NO: 121. 101. The bivalent polypeptide according to 101.
Aspect E-110: A bivalent polypeptide according to any one of aspects E-102 to E-109, wherein the at least two amino acid sequences and / or Nanobodies® are identical.
Aspect E-111:
a) SEQ ID NO: 60 to SEQ ID NO: 76
b) an amino acid sequence having only one amino acid difference from one of SEQ ID NO: 60 to SEQ ID NO: 76, preferably only two, more preferably only one;
i) the amino acid sequence has glutamine (Gln, Q) at position 105 (the position is determined by Kabat numbering);
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
A divalent polypeptide according to aspect E-100 or E-101, comprising or consisting essentially of two amino acid sequences and / or Nanobodies® selected from:
Aspect E-112: A bivalent polypeptide according to aspect E-111, wherein the two amino acid sequences and / or Nanobodies® are identical.
Aspect E-113: Aspect E-, comprising or consisting essentially of two identical amino acid sequences and / or Nanobodies® selected from one of SEQ ID NO: 60-76. 112. The bivalent polypeptide according to 112.
Aspect E-114:
a) SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76
b) only three, preferably only two and more preferably only one amino acid difference from one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75 and SEQ ID NO: 76 An amino acid sequence having
i) the amino acid sequence has glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and / or arginine (Arg, R) at position 83 (the above positions are determined by Kabat numbering; Desired),
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
A divalent polypeptide according to aspect E-100 or E-101, comprising or consisting essentially of two amino acid sequences and / or Nanobodies® selected from:
Aspect E-115:
a) SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76
b) only three, preferably only two and more preferably only one amino acid difference from one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75 and SEQ ID NO: 76 An amino acid sequence having
i) the amino acid sequence has glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and arginine (Arg, R) at position 83 (the above positions are determined by Kabat numbering) ),
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
A bivalent polypeptide according to aspect E-114, which comprises or consists essentially of two amino acid sequences and / or Nanobodies® selected from:
Aspect E-116: A bivalent polypeptide according to aspect E-114 or E-115, wherein the two amino acid sequences and / or Nanobodies® are identical.
Aspect E-117: Two identical amino acid sequences and / or Nanobodies selected from one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76 ( (Registered trademark) or consisting essentially thereof.
Aspect E-118:
a) SEQ ID NO: 65 and SEQ ID NO: 76,
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NO: 65 and SEQ ID NO: 76,
i) the amino acid sequence is aspartic acid (Asp, D) at position 54, glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and / or arginine (Arg, R) at position 83; (The position is determined by Kabat numbering),
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
A divalent polypeptide according to aspect E-100 or E-101, comprising or consisting essentially of two amino acid sequences and / or Nanobodies® selected from:
Aspect E-119:
a) SEQ ID NO: 65 and SEQ ID NO: 76,
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NO: 65 and SEQ ID NO: 76,
i) the amino acid sequence has aspartic acid (Asp, D) at position 54, glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and arginine (Arg, R) at position 83; (The above position is obtained by Kabat numbering)
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
The bivalent polypeptide according to aspect E-118, comprising or consisting essentially of two amino acid sequences and / or Nanobodies® selected from:
Aspect E-120: A bivalent polypeptide according to aspect E-118 or E-119, wherein the two amino acid sequences and / or Nanobodies® are identical.
Aspect E-121: A two-part according to aspect E-120, comprising or consisting essentially of two identical amino acid sequences and / or Nanobodies® selected from SEQ ID NO: 65 and SEQ ID NO: 76. Polyvalent polypeptide.
Aspect E-122:
a) SEQ ID NO: 146 to SEQ ID NO: 153
b) an amino acid sequence having only one, preferably two, more preferably only one amino acid difference from one of SEQ ID NOs: 146 to 153,
i) the amino acid sequence has proline (Pro, P) at position 14, arginine (Arg, R) at position 19, leucine (Leu, L) at position 20, and leucine (Leu, L) at position 108 (The above position is determined by Kabat numbering),
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
A divalent polypeptide according to aspect E-100 or E-101, comprising or consisting essentially of two amino acid sequences and / or Nanobodies® selected from:
Aspect E-123: A bivalent polypeptide according to aspect E-122, wherein the two amino acid sequences and / or Nanobodies® are identical.
Aspect E-124: Aspect E- comprising, consisting essentially of, or consisting of two identical amino acid sequences and / or Nanobodies® selected from one of SEQ ID NOs: 146-153. 123. The bivalent polypeptide according to 123.
Aspect E-125:
a) SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153,
i) the amino acid sequence comprises proline (Pro, P) at position 14, arginine (Arg, R) at position 19, leucine (Leu, L) at position 20, and leucine (Leu, L) at position 108; Arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85, and / or glutamine (Gln, Q) at position 105 (the position is determined by Kabat numbering);
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
A divalent polypeptide according to aspect E-100 or E-101, comprising or consisting essentially of two amino acid sequences and / or Nanobodies® selected from:
Aspect E-126:
a) SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153,
i) the amino acid sequence is
SEQ ID NO: 146 (this amino acid sequence preferably has glutamine (Gln, Q) at position 105);
SEQ ID NO: 147 (this amino acid sequence preferably has arginine (Arg, R) at position 83 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 148 (this amino acid sequence preferably has glutamic acid (Glu, E) at position 85 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 149 (this amino acid sequence preferably has arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 151 (this amino acid sequence preferably has arginine (Arg, R) at position 83); SEQ ID NO: 152 (this amino acid sequence preferably has glutamic acid (Glu, E) at position 85);
SEQ ID NO: 153 (this amino acid sequence preferably has arginine (Arg, R) at position 83 and glutamic acid (Glu, E) at position 85)
The amino acid sequence has a proline (Pro, P) at position 14 and an arginine (Arg, R) at position 19, as in the case of having only 3, preferably 2, and more preferably only 1 amino acid difference. Leucine (Leu, L) at position 20, Leucine (Leu, L) at position 108, arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85, and / or glutamine at position 105 (Gln, Q) (the position is determined by Kabat numbering),
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
The bivalent polypeptide according to aspect E-125, comprising or consisting essentially of two amino acid sequences and / or Nanobodies® selected from:
Aspect E-127: A bivalent polypeptide according to aspect E-125 or E-126, wherein the two amino acid sequences and / or Nanobodies® are identical.
Aspect E-128: selected from one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 147, SEQ ID NO: 149, and SEQ ID NO: 153 The bivalent polypeptide according to aspect E-127, comprising or consisting essentially of two identical amino acid sequences and / or Nanobodies®.
Aspect E-129: one or more (eg, 2, 3, 4, 5, 6, 7, 8, or 9, 10, 11, or 12) amino acid residues (SEQ ID NO: 2) has two or more amino acids with a sequence / mutation selected from the sequence / body 5 having a mutation selected from the sequence / body or sequence number 5 having a mutation selected from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp. (Registered trademark) or consisting essentially thereof.
Aspect E-130: Mutation of amino acid residues 5 having Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly108D with 5 amino acids. The bivalent polypeptide according to aspect E-129, comprising or consisting essentially of the sequence and / or Nanobody®.
Aspect E-131: One or more (eg, 2, 3, 4, 5, 6, 7, or 8) amino acid residues are Ser19R, Ile20Leu, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Aspect E-129, comprising or consisting essentially of two amino acid sequences having SEQ ID NO: 5 and / or Nanobodies®, having undergone a mutation selected from Arg105Gln and Gly54D. Bivalent polypeptide.
Aspect E-132: one or more (eg, 2, 3, 4, or 5) amino acid residues have been mutated selected from Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54D, 2 A bivalent polypeptide according to aspect E-129, comprising or consisting essentially of an amino acid sequence having SEQ ID NO: 5 and / or Nanobody®.
Aspect E-133: wherein the amino acid residue comprises two amino acid sequences having SEQ ID NO: 5 and / or Nanobody®, which have been mutated for Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54D. The bivalent polypeptide according to embodiment E-132, consisting essentially of the same.
Aspect E-134: Two, wherein one or more (eg, two, three or four) amino acid residues have been mutated selected from Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu. A bivalent polypeptide comprising or consisting essentially of an amino acid sequence having and / or Nanobody®.
Aspect E-135: One or more (eg, 2, 3, 4, 5, 6, or 7) amino acid residues are from Ala14Pro, Ser19Arg, Ile20Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu. A bivalent polypeptide comprising or consisting essentially of two amino acid sequences having SEQ ID NO: 5 and / or Nanobodies®, having undergone a selected mutation.
Aspect E-136:
The amino acid residue is
Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Gly78Leu, Ala83Arg, Asp85Glu, and Arg105Gln,
Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54Asp,
Gly54Asp,
Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Ala83Arg,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Asp85Glu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Arg105Gln,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Asp85Glu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Arg105Gln,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Asp85Glu, and Arg105Gln, or
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, Asp85Glu, and Arg105Gln
A bivalent polypeptide according to aspect E-129, comprising or consisting essentially of two amino acid sequences having SEQ ID NO: 5 and / or Nanobodies®, which have been mutated.
Aspect E-137:
a) SEQ ID NO: 138 to SEQ ID NO: 141 and SEQ ID NO: 154 to SEQ ID NO: 157;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 138 to 141 and 154 to 157,
i) the amino acid sequence has aspartic acid (Asp, D) at position 1 (the position is determined by Kabat numbering);
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
A bivalent polypeptide according to aspect E-100 or E-101, comprising or consisting essentially of at least one amino acid sequence and / or Nanobody® selected from the group consisting of:
Aspect E-138: Comprising or comprising at least one amino acid sequence and / or Nanobody® selected from one of SEQ ID NOs: 138-141 and SEQ ID NOs: 154-157. A bivalent polypeptide according to aspect E-137, wherein
Embodiment E-139: A divalent poly comprising, or consisting essentially of, at least one amino acid sequence having SEQ ID NO: 5 and / or Nanobody®, wherein the glutamic acid at position 1 has been changed to aspartic acid. peptide.
Aspect E-140: A divalent poly comprising, or consisting essentially of, at least one amino acid sequence having SEQ ID NO: 62 and / or Nanobody®, wherein glutamic acid at position 1 has been changed to aspartic acid. peptide.
Aspect E-141: A divalent poly comprising or consisting essentially of at least one amino acid sequence having SEQ ID NO: 65 and / or Nanobody®, wherein glutamic acid at position 1 has been changed to aspartic acid. peptide.
Aspect E-142: A divalent poly comprising or consisting essentially of at least one amino acid sequence having SEQ ID NO: 76 and / or Nanobody®, wherein glutamic acid at position 1 has been changed to aspartic acid. peptide.
Aspect E-143: Divalent poly comprising or consisting essentially of at least one amino acid sequence having SEQ ID NO: 75 and / or Nanobody®, wherein glutamic acid at position 1 has been changed to aspartic acid peptide.
Aspect E-144: A divalent poly comprising, or consisting essentially of, at least one amino acid sequence having SEQ ID NO: 147 and / or Nanobody®, wherein glutamic acid at position 1 has been changed to aspartic acid. peptide.
Aspect E-145: A divalent poly comprising, or consisting essentially of, at least one amino acid sequence having SEQ ID NO: 149 and / or Nanobody®, wherein the glutamic acid at position 1 is changed to aspartic acid. peptide.
Aspect E-146: A divalent poly comprising or consisting essentially of at least one amino acid sequence having SEQ ID NO: 153 and / or Nanobody®, wherein glutamic acid at position 1 has been changed to aspartic acid. peptide.
Aspect E-147: One or more (eg, 2, 3, 4, 5, 6, 7, 8, or 9, 10, 11, or 12) amino acid residues , Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and a mutation in which the amino acid at the amino acid position Glu54Asp has a mutation selected from the following: A bivalent polypeptide comprising or consisting essentially of at least one amino acid sequence and / or Nanobody® having the number 5.
Aspect E-148: Amino acid residue has been mutated to amino acid at position Gal at amino acid Gal at amino acid position Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54D. The bivalent polypeptide according to aspect E-147, which comprises, or consists essentially of, at least one amino acid sequence and / or Nanobody® having an altered SEQ ID NO: 5.
Aspect E-149: One or more amino acid residues selected from Ser19R, Ile20Leu, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54D (eg, 2, 3, 4, 5, 6) , 7 or 8), wherein at least one amino acid sequence having SEQ ID NO: 5 and / or Nanobody® having glutamic acid at position 1 changed to aspartic acid. Or a bivalent polypeptide according to aspect E-147, consisting essentially of, or consisting of,
Aspect E-150: one or more (eg, 2, 3, 4, or 5) amino acid residues have a mutation selected from Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54D; Aspect E-147, wherein the glutamic acid in position 1 is changed to aspartic acid, comprising or consisting essentially of at least one amino acid sequence having SEQ ID NO: 5 and / or Nanobody®. Bivalent polypeptide.
Aspect E-151: At least one having SEQ ID NO: 5, wherein the amino acid residue has been mutated for Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54D and glutamic acid at position 1 has been changed to aspartic acid The divalent polypeptide according to aspect E-150, comprising or consisting essentially of an amino acid sequence and / or Nanobody®.
Aspect E-152: wherein the amino acid residue has one or more (eg, 2, 3, or 4) mutations selected from Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu, and glutamic acid at position 1 has A bivalent polypeptide comprising or consisting essentially of at least one amino acid sequence having SEQ ID NO: 5 and / or Nanobody®, which has been changed to aspartic acid.
Aspect E-153: One or more amino acid residues selected from Ala14Pro, Ser19Arg, Ile20Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu (eg, 2, 3, 4, 5, 6, or 7). Comprising at least one amino acid sequence having SEQ ID NO: 5 and / or Nanobody®, wherein the glutamic acid at position 1 has been changed to aspartic acid. Divalent polypeptide.
Aspect E-154:
The amino acid residue is
Glu1Asp,
Glu1Asp, Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Glu1Asp, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Glu1Asp, Gly78Leu, Ala83Arg, Asp85Glu, and Arg105Gln;
Glu1Asp, Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Glu1Asp, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Glu1Asp, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54Asp,
Glu1Asp and Gly54Asp,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu, Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Ala83Arg,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Asp85Glu,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Arg105Gln,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Asp85Glu,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Arg105Gln,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Asp85Glu, and Arg105Gln, or
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, Asp85Glu, and Arg105Gln
The bivalent polypeptide according to aspect E-147, comprising or consisting essentially of at least one amino acid sequence having SEQ ID NO: 5 and / or Nanobody®, mutated to:
Aspect E-155: Three amino acid sequences according to any one of Aspects A-1 to A-29, Aspects C-1 to C-38, and Aspects W-1 to W-38, and / or Aspects Comprising or consisting essentially of Nanobody (R) according to any one of B-1 to B-18, Aspects D-1 to D-38, and Aspects X-1 to X-38, The trivalent polypeptide according to aspect E-7.
Aspect E-156: Trivalent polypeptide according to aspect E-155, wherein the three amino acid sequences and / or Nanobodies® are identical.
Aspect E-157:
a) SEQ ID NO: 102,
b) a stretch of amino acid residues having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 102,
i) the amino acid residue stretch has aspartic acid (Asp, D) at position 6 (position 54 determined by Kabat numbering),
ii) the amino acid sequence comprising the amino acid residue stretch has the same, substantially the same or higher affinity as compared to the amino acid sequence comprising the amino acid residue stretch having no three, two or one amino acid difference ( The affinity is as measured by surface plasmon resonance) and binds to protein F of hRSV and / or the amino acid sequence comprising said stretch of amino acid residues differs by three, two or one amino acid An amino acid selected from an amino acid residue stretch that has the same, approximately the same, or a higher potency (as defined herein) as compared to an amino acid sequence comprising the above amino acid residue stretch without Or three amino acid sequences comprising at least one residue stretch and / or Nanobody® Luo consisting essentially trivalent polypeptide according to aspect E-155 or E-156.
Aspect E-158:
a) SEQ ID NO: 102,
b) a stretch of amino acid residues having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 102,
i) the amino acid residue stretch has aspartic acid (Asp, D) at position 6 (position 54 determined by Kabat numbering),
ii) the amino acid sequence comprising the amino acid residue stretch has the same, substantially the same or higher affinity as compared to the amino acid sequence comprising the amino acid residue stretch having no three, two or one amino acid difference ( The affinity is as measured by surface plasmon resonance) and binds to protein F of hRSV and / or the amino acid sequence comprising said stretch of amino acid residues differs by three, two or one amino acid Selected from amino acid residue stretches that have the same, approximately the same, or higher potency (as defined herein) as compared to an amino acid sequence comprising the amino acid residue stretch without At least one amino acid residue stretch,
At least one stretch,
c) SEQ ID NO: 98;
d) An amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 98, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (as said affinity is measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference the amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; An amino acid residue stretch, which shall have about the same or higher potency (as defined herein);
e) SEQ ID NO: 121, and
f) an amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 121, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (such as measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference an amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Select from stretches of amino acid residues that should have about the same or higher potency (as defined herein). The amino acid residue stretch comprising or consisting essentially of three amino acid sequences and / or Nanobodies®, corresponding to one of a) and b), is always the amino acid sequence of the invention. Wherein the second amino acid residue stretch is as selected from one of c), d), e) and f). 156. Trivalent polypeptide according to 156.
Aspect E-159:
a) SEQ ID NO: 98, and
b) an amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 98, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (as said affinity is measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference the amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Select from stretches of amino acid residues that should have about the same or higher potency (as defined herein) Is, at least one amino acid residue stretch,
c) SEQ ID NO: 102, and
d) a stretch of amino acid residues having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 102,
i) the amino acid residue stretch has aspartic acid (Asp, D) at position 6 (position 54 determined by Kabat numbering),
ii) the amino acid sequence comprising the amino acid residue stretch has the same, substantially the same or higher affinity as compared to the amino acid sequence comprising the amino acid residue stretch having no three, two or one amino acid difference ( The affinity is as measured by surface plasmon resonance) and binds to protein F of hRSV and / or the amino acid sequence comprising said stretch of amino acid residues differs by three, two or one amino acid Selected from the group consisting of amino acid residue stretches that have the same, approximately the same, or higher potency (as defined herein) as compared to an amino acid sequence comprising the above amino acid residue stretches without A second stretch of amino acid residues,
e) SEQ ID NO: 121, and
f) an amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 121, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (such as measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference an amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Stretches of amino acid residues which should be of about the same or higher potency (as defined herein). E-155 or E-156, comprising or consisting essentially of three amino acid sequences and / or Nanobodies®, including a third amino acid residue stretch selected from the group: Trivalent polypeptide.
Aspect E-160: Trivalent polypeptide according to aspect E-155 or E-156, comprising or consisting essentially of a three amino acid sequence comprising at least SEQ ID NO: 102 and / or Nanobody®. .
Aspect E-161: at least SEQ ID NO: 102,
a) SEQ ID NO: 98,
b) an amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 98, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (as said affinity is measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference the amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; An amino acid residue stretch, which shall have about the same or higher potency (as defined herein);
c) SEQ ID NO: 121, and
d) An amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 121, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (as said affinity is measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference the amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Select from stretches of amino acid residues that should have about the same or higher potency (as defined herein). Aspect E-155 or E-156 comprising or consisting essentially of three amino acid sequences and / or Nanobodies®, including at least one stretch of amino acid residues (CDR sequences). The trivalent polypeptide of the description.
Aspect E-162: at least SEQ ID NO: 102;
a) SEQ ID NO: 98,
b) an amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 98, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (as said affinity is measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference the amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Select from stretches of amino acid residues that should have about the same or higher potency (as defined herein) Is a CDR1 sequence selected from at least one amino acid residue stretch,
c) SEQ ID NO: 121, and
d) An amino acid residue stretch having only 3, preferably 2, and more preferably only 1 amino acid difference from SEQ ID NO: 121, wherein the amino acid sequence containing the amino acid residue stretch is 3, 2 Or with the same, about the same, or a higher affinity (as said affinity is measured by surface plasmon resonance) compared to an amino acid sequence comprising said amino acid residue stretch without one amino acid difference the amino acid sequence that binds to protein F of hRSV and / or comprises said stretch of amino acid residues is the same as said amino acid sequence comprising said stretch of amino acid residues without three, two or one amino acid difference; Select from stretches of amino acid residues that should have about the same or higher potency (as defined herein). It is the and a CDR3 sequence comprises or three amino acid sequences and / or Nanobody (TM), consists essentially or therefrom, trivalent polypeptide according to aspect E-155 or E-156.
Aspect E-163: Three amino acid sequence and / or Nanobody® comprising at least SEQ ID NO: 102 and at least one amino acid residue stretch (CDR sequence) selected from SEQ ID NO: 98 and SEQ ID NO: 121 A trivalent polypeptide according to aspect E-155 or E-156, comprising or consisting essentially of:
Aspect E-164: Aspect E-155 or E- comprising, or consisting essentially of, three amino acid sequences and / or Nanobodies®, including SEQ ID NO: 98, SEQ ID NO: 102, and SEQ ID NO: 121. 156. Trivalent polypeptide according to 156.
Aspect E-165: Trivalent polypeptide according to any one of aspects E-157 to E-164, wherein the at least three amino acid sequences and / or Nanobodies® are identical.
Aspect E-166:
a) SEQ ID NO: 60 to SEQ ID NO: 76
b) an amino acid sequence having only one amino acid difference from one of SEQ ID NO: 60 to SEQ ID NO: 76, preferably only two, more preferably only one;
i) the amino acid sequence has glutamine (Gln, Q) at position 105 (the position is determined by Kabat numbering);
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
A trivalent polypeptide according to aspect E-155 or E-156, comprising or consisting essentially of three amino acid sequences and / or Nanobodies® selected from:
Aspect E-167: Trivalent polypeptide according to aspect E-166, wherein said three amino acid sequences and / or Nanobodies® are identical.
Aspect E-168: Aspect E-, comprising or consisting essentially of three identical amino acid sequences and / or Nanobodies® selected from one of SEQ ID NO: 60-SEQ ID NO: 76. 167. The trivalent polypeptide according to 167.
Aspect E-169:
a) SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76
b) only three, preferably only two and more preferably only one amino acid difference from one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75 and SEQ ID NO: 76 I) the amino acid sequence has glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and arginine (Arg, R) at position 83 (as described above); Is determined by Kabat numbering),
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
A trivalent polypeptide according to aspect E-155 or E-156, comprising or consisting essentially of three amino acid sequences and / or Nanobodies® selected from:
Aspect E-170:
a) SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76
b) only three, preferably only two and more preferably only one amino acid difference from one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75 and SEQ ID NO: 76 An amino acid sequence having
i) the amino acid sequence has glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and arginine (Arg, R) at position 83 (the above positions are determined by Kabat numbering) ),
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
A trivalent polypeptide according to aspect E-169, comprising or consisting essentially of three amino acid sequences and / or Nanobodies® selected from:
Aspect E-171: Trivalent polypeptide according to aspect E-169 or E-170, wherein the three amino acid sequences and / or Nanobodies® are identical.
Aspect E-172: Three identical amino acid sequences and / or Nanobodies selected from one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76 ( (Registered trademark) or consisting essentially thereof.
Aspect E-173:
a) SEQ ID NO: 65 and SEQ ID NO: 76,
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NO: 65 and SEQ ID NO: 76,
i) the amino acid sequence is aspartic acid (Asp, D) at position 54, glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and / or arginine (Arg, R) at position 83; (The position is determined by Kabat numbering),
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
A trivalent polypeptide according to aspect E-155 or E-156, comprising or consisting essentially of three amino acid sequences and / or Nanobodies® selected from:
Aspect E-174: a) SEQ ID NO: 65 and SEQ ID NO: 76,
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NO: 65 and SEQ ID NO: 76,
i) the amino acid sequence has aspartic acid (Asp, D) at position 54, glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and arginine (Arg, R) at position 83; (The above position is obtained by Kabat numbering)
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
A trivalent polypeptide according to aspect E-173, comprising or consisting essentially of three amino acid sequences and / or Nanobodies® selected from:
Aspect E-175: Trivalent polypeptide according to aspect E-173 or E-174, wherein the three amino acid sequences and / or Nanobodies® are identical.
Aspect E-176: A triad according to aspect E-175, comprising or consisting essentially of three identical amino acid sequences and / or Nanobodies® selected from SEQ ID NO: 65 and SEQ ID NO: 76. Polyvalent polypeptide.
Aspect E-177:
a) SEQ ID NO: 146 to SEQ ID NO: 153
b) an amino acid sequence having only one, preferably two, more preferably only one amino acid difference from one of SEQ ID NOs: 146 to 153,
i) the amino acid sequence has proline (Pro, P) at position 14, arginine (Arg, R) at position 19, leucine (Leu, L) at position 20, and leucine (Leu, L) at position 108 (The above position is determined by Kabat numbering),
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
A trivalent polypeptide according to aspect E-155 or E-156, comprising or consisting essentially of three amino acid sequences and / or Nanobodies® selected from:
Aspect E-178: Trivalent polypeptide according to aspect E-177, wherein the three amino acid sequences and / or Nanobodies® are identical.
Aspect E-179: Aspect E-, which comprises or consists essentially of three identical amino acid sequences and / or Nanobodies® selected from one of SEQ ID NO: 146 to SEQ ID NO: 153. 178. Trivalent polypeptide according to 178.
Aspect E-180:
a) SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153,
i) the amino acid sequence comprises proline (Pro, P) at position 14, arginine (Arg, R) at position 19, leucine (Leu, L) at position 20, and leucine (Leu, L) at position 108; Arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85, and / or glutamine (Gln, Q) at position 105 (the position is determined by Kabat numbering);
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
A trivalent polypeptide according to aspect E-155 or E-156, comprising or consisting essentially of three amino acid sequences and / or Nanobodies® selected from:
Aspect E-181:
a) SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153,
i) the amino acid sequence is
SEQ ID NO: 146 (this amino acid sequence preferably has glutamine (Gln, Q) at position 105);
SEQ ID NO: 147 (this amino acid sequence preferably has arginine (Arg, R) at position 83 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 148 (this amino acid sequence preferably has glutamic acid (Glu, E) at position 85 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 149 (this amino acid sequence preferably has arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 151 (this amino acid sequence preferably has arginine (Arg, R) at position 83);
SEQ ID NO: 152 (this amino acid sequence preferably has glutamic acid (Glu, E) at position 85);
SEQ ID NO: 153 (this amino acid sequence preferably has arginine (Arg, R) at position 83 and glutamic acid (Glu, E) at position 85)
The amino acid sequence has a proline (Pro, P) at position 14 and an arginine (Arg, R) at position 19, as in the case of having only 3, preferably 2, and more preferably only 1 amino acid difference. Leucine (Leu, L) at position 20, Leucine (Leu, L) at position 108, arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85, and / or glutamine at position 105 (Gln, Q) (the position is determined by Kabat numbering),
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
A trivalent polypeptide according to aspect E-180, which comprises or consists essentially of three amino acid sequences and / or Nanobodies® selected from:
Aspect E-182: Trivalent polypeptide according to aspect E-180 or E-181, wherein the three amino acid sequences and / or Nanobodies® are identical.
Aspect E-183: Three identical sequences selected from SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 147, SEQ ID NO: 149, and SEQ ID NO: 153 The trivalent polypeptide according to aspect E-182, comprising or consisting essentially of an amino acid sequence and / or Nanobody®.
Aspect E-184: wherein the amino acid residue is selected from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and one or more Gly54Asp, e.g. 3 amino acid sequences having SEQ ID NO: 5 and / or Nanobodies (4, 5, 6, 7, 8, or 9, 10, 11, or 12) mutated (Registered trademark) or consisting essentially thereof.
Aspect E-185: Three amino acid residues having the following sequence: Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and three amino acids having the following mutations: Gly108D. The trivalent polypeptide according to aspect E-184, comprising or consisting essentially of sequence and / or Nanobody®.
Aspect E-186: One or more amino acid residues selected from Ser19R, Ile20Leu, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54D (eg, two, three, four, five, six) According to aspect E-184, comprising or consisting essentially of three, 7 or 8) mutated amino acid sequences having SEQ ID NO: 5 and / or Nanobodies®. Trivalent polypeptide.
Aspect E-187: wherein the amino acid residue has one or more (eg, 2, 3, 4, or 5) mutations selected from Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54D, 3 A trivalent polypeptide according to aspect E-184, comprising or consisting essentially of an amino acid sequence having SEQ ID NO: 5 and / or Nanobody®.
Aspect E-188: Three or more amino acid sequences having SEQ ID NO: 5 and / or Nanobody®, wherein the amino acid residue has been mutated for Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54D. A trivalent polypeptide according to aspect E-187, consisting essentially of the same.
Aspect E-189: 3 SEQ ID NO: 5 wherein one or more (eg, 2, 3 or 4) amino acid residues have been mutated selected from Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu. A trivalent polypeptide comprising or consisting essentially of an amino acid sequence having and / or Nanobody®.
Aspect E-190: One or more (eg, 2, 3, 4, 5, 6, or 7) amino acid residues from Ala14Pro, Ser19Arg, Ile20Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu. 3. A trivalent polypeptide comprising or consisting essentially of three amino acid sequences having SEQ ID NO: 5 and / or Nanobodies®, having undergone a selected mutation.
Aspect E-191:
The amino acid residue is
Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Gly78Leu, Ala83Arg, Asp85Glu, and Arg105Gln,
Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54Asp,
Gly54Asp, Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Ala83Arg,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Asp85Glu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Arg105Gln,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Asp85Glu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Arg105Gln,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Asp85Glu, and Arg105Gln, or
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, Asp85Glu, and Arg105Gln
A trivalent polypeptide according to aspect E-184, comprising or consisting essentially of three amino acid sequences having SEQ ID NO: 5 and / or Nanobodies®, which have been mutated.
Aspect E-192: Three trivalent polypeptides comprising or consisting essentially of an amino acid sequence having SEQ ID NO: 62 and / or Nanobody®.
Aspect E-193: Trivalent polypeptide comprising or consisting essentially of three amino acid sequences having SEQ ID NO: 65 and / or Nanobodies®.
Aspect E-194: Trivalent polypeptide comprising or consisting essentially of three amino acid sequences having SEQ ID NO: 76 and / or Nanobodies®.
Aspect E-195: Three trivalent polypeptides comprising or consisting essentially of an amino acid sequence having SEQ ID NO: 75 and / or Nanobody®.
Aspect E-196: Three trivalent polypeptides comprising or consisting essentially of an amino acid sequence having SEQ ID NO: 147 and / or Nanobody®.
Aspect E-197: Three trivalent polypeptides comprising or consisting essentially of an amino acid sequence having SEQ ID NO: 149 and / or Nanobody®.
Aspect E-198: Three trivalent polypeptides comprising or consisting essentially of an amino acid sequence having SEQ ID NO: 153 and / or Nanobody®.
Aspect E-199:
a) SEQ ID NO: 138 to SEQ ID NO: 141 and SEQ ID NO: 154 to SEQ ID NO: 157;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 138 to 141 and 154 to 157,
i) the amino acid sequence has aspartic acid (Asp, D) at position 1 (the position is determined by Kabat numbering);
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
A trivalent polypeptide according to aspect E-155 or E-156, comprising or consisting essentially of at least one amino acid sequence and / or Nanobody® selected from the group consisting of:
Aspect E-200: Comprising or comprising at least one amino acid sequence and / or Nanobody® selected from one of SEQ ID NOs: 138-141 and SEQ ID NOs: 154-157. A trivalent polypeptide according to aspect E-199, wherein
Aspect E-201: Trivalent poly comprising or consisting essentially of at least one amino acid sequence having SEQ ID NO: 5 and / or Nanobody®, wherein glutamic acid at position 1 has been changed to aspartic acid peptide.
Aspect E-202: Trivalent poly comprising or consisting essentially of at least one amino acid sequence having SEQ ID NO: 62 and / or Nanobody®, wherein glutamic acid at position 1 has been changed to aspartic acid peptide.
Aspect E-203: Trivalent poly comprising or consisting essentially of at least one amino acid sequence having SEQ ID NO: 65 and / or Nanobody®, wherein glutamic acid at position 1 has been changed to aspartic acid peptide.
Aspect E-204: Trivalent poly comprising or consisting essentially of at least one amino acid sequence having SEQ ID NO: 76 and / or Nanobody®, wherein glutamic acid at position 1 has been changed to aspartic acid peptide.
Aspect E-205: Trivalent poly comprising or consisting essentially of at least one amino acid sequence having SEQ ID NO: 75 and / or Nanobody®, wherein glutamic acid at position 1 has been changed to aspartic acid peptide.
Aspect E-206: Trivalent poly comprising or consisting essentially of at least one amino acid sequence having SEQ ID NO: 147 and / or Nanobody®, wherein glutamic acid at position 1 has been changed to aspartic acid peptide.
Aspect E-207: Trivalent poly comprising or consisting essentially of at least one amino acid sequence having SEQ ID NO: 149 and / or Nanobody®, wherein glutamic acid at position 1 has been changed to aspartic acid peptide.
Aspect E-208: Trivalent poly comprising, or consisting essentially of, at least one amino acid sequence having SEQ ID NO: 153 and / or Nanobody®, wherein glutamic acid at position 1 has been changed to aspartic acid. peptide.
Aspect E-209: One or more (eg, 2, 3, 4, 5, 6, 7, 8, or 9, 10, 11, or 12) amino acid residues , Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and a mutation in which the amino acid at the amino acid position Glu54Asp has a mutation selected from the following: A trivalent polypeptide comprising, or consisting essentially of, at least one amino acid sequence and / or Nanobody® having the number 5.
Aspect E-210: Amino acid residue has been mutated at amino acid position Palminate at amino acid position Val1Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54D. The trivalent polypeptide according to aspect E-209, which comprises, or consists essentially of, at least one amino acid sequence and / or Nanobody® having an altered SEQ ID NO: 5.
Embodiment E-211: wherein one or more (eg, 2, 3, 4, 5, 6, 7, or 8) amino acid residues are Ser19R, Ile20Leu, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Contains at least one amino acid sequence having SEQ ID NO: 5 and / or Nanobody®, which has a mutation selected from Arg105Gln, and Gly54D, wherein glutamic acid at position 1 has been changed to aspartic acid. Or consisting essentially of, or consisting of, the embodiment E-209.
Aspect E-212: one or more (eg, 2, 3, 4, or 5) amino acid residues have a mutation selected from Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54D; Aspect E-209, wherein the glutamic acid at position 1 is changed to aspartic acid, comprising or consisting essentially of at least one amino acid sequence having SEQ ID NO: 5 and / or Nanobody®. Trivalent polypeptide.
Aspect E-213: At least one having SEQ ID NO: 5, wherein the amino acid residue has been mutated for Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54D, and wherein glutamic acid at position 1 has been changed to aspartic acid. The trivalent polypeptide according to aspect E-212, which comprises or consists essentially of the amino acid sequence and / or Nanobody®.
Aspect E-214:
The amino acid residue is
Glu1Asp,
Glu1Asp, Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Glu1Asp, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Glu1Asp, Gly78Leu, Ala83Arg, Asp85Glu, and Arg105Gln;
Glu1Asp, Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Glu1Asp, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Glu1Asp, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54Asp,
Glu1Asp and Gly54Asp,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Ala83Arg;
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Asp85Glu,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Arg105Gln,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Asp85Glu,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Arg105Gln,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Asp85Glu, and Arg105Gln, or
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, Asp85Glu, and Arg105Gln
The trivalent polypeptide according to aspect E-209, comprising or consisting essentially of at least one amino acid sequence having SEQ ID NO: 5 and / or Nanobody®, mutated to:
Aspect E-215: The following polypeptide:
a) SEQ ID NO: 77 to SEQ ID NO: 79 and SEQ ID NO: 158;
b) a polypeptide which has no more than 3, preferably no more than 2, and more preferably no more than one amino acid difference from one of SEQ ID NOs: 77-79 and 158,
i) the amino acid sequence or Nanobody (registered trademark) contained in the polypeptide is glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, arginine (Arg, R) at position 83; And / or has glutamic acid (Glu, E) at position 85 (the position is determined by Kabat numbering),
ii) the polypeptide has the same, about the same, or a higher affinity as compared to a polypeptide having no three, two or one amino acid difference (as the affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said polypeptide has the same, about the same, or higher potency (as compared to a polypeptide without three, two or one amino acid difference) Polypeptide (as defined herein)
A trivalent polypeptide having directionality and / or specifically binding to protein F of hRSV, selected from the group consisting of:
Aspect E-216: The following polypeptide:
a) SEQ ID NO: 77 to SEQ ID NO: 79 and SEQ ID NO: 158;
b) a polypeptide which has no more than 3, preferably no more than 2, and more preferably no more than one amino acid difference from one of SEQ ID NOs: 77-79 and 158,
i) the amino acid sequence or Nanobody (registered trademark) contained in the polypeptide is glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, arginine (Arg, R) at position 83; And has glutamic acid (Glu, E) at position 85 (the position is determined by Kabat numbering),
ii) the polypeptide has the same, about the same, or a higher affinity as compared to a polypeptide having no three, two or one amino acid difference (as the affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said polypeptide has the same, about the same, or higher potency (as compared to a polypeptide without three, two or one amino acid difference) Polypeptide (as defined herein)
The trivalent polypeptide according to aspect E-215, which is selected from:
Aspect E-217: The following polypeptide:
a) SEQ ID NO: 78 and SEQ ID NO: 79,
b) a polypeptide having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NO: 78 and SEQ ID NO: 79,
i) The amino acid sequence or Nanobody (registered trademark) contained in the polypeptide is aspartic acid (Asp, D) at position 54, glutamine (Gln, Q) at position 105, and leucine (Leu, L) at position 78 Having arginine (Arg, R) at position 83 and / or glutamic acid (Glu, E) at position 85 (the position is determined by Kabat numbering);
ii) the polypeptide has the same, about the same, or a higher affinity as compared to a polypeptide having no three, two or one amino acid difference (as the affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said polypeptide has the same, about the same, or higher potency (as compared to a polypeptide without three, two or one amino acid difference) Polypeptide (as defined herein)
A trivalent polypeptide having directionality and / or specifically binding to protein F of hRSV, selected from the group consisting of:
Aspect E-218: The following polypeptide:
a) SEQ ID NO: 78 and SEQ ID NO: 79,
b) a polypeptide having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NO: 78 and SEQ ID NO: 79,
i) the amino acid sequence or Nanobody (registered trademark) contained in the polypeptide is aspartic acid (Asp, D) at position 54, glutamine (Gln, Q) at position 105, and leucine (Leu, L) at position 78 Arginine (Arg, R) at position 83 and glutamic acid (Glu, E) at position 85 (the positions are determined by Kabat numbering); ii) the polypeptide is three, two or one Binds to protein F of hRSV with the same, about the same, or a higher affinity (where the affinity is as measured by surface plasmon resonance) compared to a polypeptide having no two amino acid differences; and And / or the same, about the same, or higher potency as compared to a polypeptide that does not have three, two or one amino acid difference (the present invention). Polypeptide as defined in the detailed description)
The trivalent polypeptide according to aspect E-217, which is selected from:
Aspect E-219: The following polypeptide:
a) SEQ ID NO: 159 to SEQ ID NO: 161,
b) a polypeptide having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 159 to 161;
i) the amino acid sequence or Nanobody (registered trademark) contained in the polypeptide is proline (Pro, P) at position 14, arginine (Arg, R) at position 19, leucine (Leu, L) at position 20, And leucine (Leu, L) at position 108 (the position is determined by Kabat numbering),
ii) the polypeptide has the same, about the same, or a higher affinity as compared to a polypeptide having no three, two or one amino acid difference (as the affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said polypeptide has the same, about the same, or higher potency (as compared to a polypeptide without three, two or one amino acid difference) Polypeptide (as defined herein)
A trivalent polypeptide having directionality and / or specifically binding to protein F of hRSV, selected from the group consisting of:
Aspect E-220: The following polypeptide:
a) SEQ ID NO: 159 to SEQ ID NO: 161,
b) a polypeptide having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 159 to 161;
i) the amino acid sequence or Nanobody (registered trademark) contained in the polypeptide is proline (Pro, P) at position 14, arginine (Arg, R) at position 19, leucine (Leu, L) at position 20, And leucine (Leu, L) at position 108, arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85, and / or glutamine (Gln, Q) at position 105 ( The above position is obtained by Kabat numbering),
ii) the polypeptide has the same, about the same, or a higher affinity as compared to a polypeptide having no three, two or one amino acid difference (as the affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said polypeptide has the same, about the same, or higher potency (as compared to a polypeptide without three, two or one amino acid difference) Polypeptide (as defined herein)
The trivalent polypeptide according to aspect E-219, which is selected from:
Aspect E-221: The following polypeptide:
a) SEQ ID NO: 159 to SEQ ID NO: 161,
b) a polypeptide having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 159 to 161;
i) the polypeptide is
SEQ ID NO: 159 (amino acid sequence or Nanobody® included in the polypeptide preferably has arginine (Arg, R) at position 83 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 160 (the amino acid sequence or Nanobody® included in the above polypeptide is preferably arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85 and glutamine (Gln, SEQ ID NO: 161 (amino acid sequence or Nanobody® included in the polypeptide preferably has arginine (Arg, R) at position 83 and glutamic acid (Glu, E) at position 85 )
And the amino acid sequence or Nanobody (R) encompassed within the polypeptide, as in the case of having only 3, preferably 2, and more preferably only 1 amino acid difference. , P), arginine (Arg, R) at position 19, leucine (Leu, L) at position 20, leucine (Leu, L) at position 108, arginine (Arg, R) at position 83, and glutamic acid at position 85 (Glu, E), and / or glutamine (Gln, Q) at position 105 (the position is determined by Kabat numbering),
ii) the polypeptide has the same, about the same, or a higher affinity as compared to an amino acid sequence that does not have three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance) That bind to protein F of hRSV and / or wherein said polypeptide has the same, approximately the same, or higher potency (as compared to an amino acid sequence having no three, two or one amino acid difference). Polypeptide (as defined herein)
The trivalent polypeptide according to aspect E-219 or E-220, which is selected from:
Aspect E-222: one or more (eg, 2, 3, 4, 5, 6, 7, 8, or 9, 10, 11, or 12) amino acid residues In at least one (preferably two, more preferably all three) Nanobodies® forming part of SEQ ID NO: 53, Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, A trivalent polypeptide comprising or consisting essentially of SEQ ID NO: 53, mutated selected from Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp.
Aspect E-223: In at least one (preferably two, more preferably all three) Nanobodies® forming part of SEQ ID NO: 53, the amino acid residue is Val5Leu, Ala14Pro, Ser19R, Ile20Leu. A trivalent polypeptide comprising or consisting essentially of SEQ ID NO: 53, mutated from Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54D.
Aspect E-224: In at least one (preferably two, more preferably all three) Nanobodies® forming part of SEQ ID NO: 53, one or more (eg, two, three, SEQ ID NO: 53 wherein 4, 5, 6, 7, or 8) amino acid residues have undergone a mutation selected from Ser19R, Ile20Leu, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54D. A trivalent polypeptide comprising or consisting essentially of:
Aspect E-225: In at least one (preferably two, more preferably all three) Nanobodies® forming part of SEQ ID NO: 53, one or more (eg, two, three, A trivalent polypeptide comprising or consisting essentially of SEQ ID NO: 53, wherein four or five) amino acid residues are mutated selected from Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54D.
Aspect E-226: In at least one (preferably two, more preferably all three) Nanobodies® forming part of SEQ ID NO: 53, wherein the amino acid residue is Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln. And a trivalent polypeptide comprising or consisting essentially of SEQ ID NO: 53, mutated with Gly54D.
Aspect E-227: In at least one (preferably two, more preferably all three) Nanobodies® forming a part of SEQ ID NO: 53, one or more (eg, two, three or A trivalent polypeptide comprising or consisting essentially of SEQ ID NO: 53, wherein 4) the amino acid residues have been mutated selected from Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu.
Aspect E-228: In at least one (preferably two, more preferably all three) Nanobodies® forming part of SEQ ID NO: 53, one or more (eg, two, three, SEQ ID NO: 53, wherein the 4, 5, 6, or 7) amino acid residues have a mutation selected from Ala14Pro, Ser19Arg, Ile20Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu, or A trivalent polypeptide consisting essentially of this.
Aspect E-229: In at least one (preferably two, more preferably all three) Nanobodies® forming part of SEQ ID NO: 53, the amino acid residue is:
Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Gly78Leu, Ala83Arg, Asp85Glu, and Arg105Gln,
Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54Asp,
Gly54Asp,
Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Ala83Arg,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Asp85Glu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Arg105Gln,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Asp85Glu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Arg105Gln,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Asp85Glu, and Arg105Gln, or
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, Asp85Glu, and Arg105Gln
A trivalent polypeptide comprising or consisting essentially of SEQ ID NO: 53, mutated to:
Aspect E-230: A trivalent polypeptide comprising or consisting essentially of SEQ ID NO: 77.
Aspect E-231: Trivalent polypeptide comprising or consisting essentially of SEQ ID NO: 78.
Aspect E-232: Trivalent polypeptide comprising or consisting essentially of SEQ ID NO: 79.
Aspect E-233: Trivalent polypeptide comprising, or consisting essentially of, one of SEQ ID NOs: 159-161.
Aspect E-234: The following polypeptide:
a) SEQ ID NO: 142 to SEQ ID NO: 145 and SEQ ID NO: 162 to SEQ ID NO: 165;
b) a polypeptide having an amino acid difference of no more than 3, preferably no more than 2, and more preferably no more than one of SEQ ID NOs: 142 to 145 and SEQ ID NOs: 162 to 165,
i) the first amino acid sequence or Nanobody® included in the polypeptide has aspartic acid (Asp, D) at position 1;
ii) the polypeptide has the same, about the same, or a higher affinity as compared to a polypeptide having no three, two or one amino acid difference (as the affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said polypeptide has the same, about the same, or higher potency (as compared to a polypeptide without three, two or one amino acid difference) Polypeptide (as defined herein)
A trivalent polypeptide having directionality and / or specifically binding to protein F of hRSV, selected from the group consisting of:
Aspect E-235 A trivalent polypeptide comprising or consisting essentially of SEQ ID NO: 53, wherein the glutamic acid at position 1 is changed to aspartic acid.
Aspect E-236. A trivalent polypeptide comprising or consisting essentially of SEQ ID NO: 77, wherein the glutamic acid at position 1 has been changed to aspartic acid.
Aspect E-237. Trivalent polypeptide comprising or consisting essentially of SEQ ID NO: 78, wherein the glutamic acid at position 1 has been changed to aspartic acid.
Aspect E-238. Trivalent polypeptide comprising or consisting essentially of SEQ ID NO: 79, wherein the glutamic acid at position 1 has been changed to aspartic acid.
Aspect E-239. Trivalent polypeptide comprising or consisting essentially of SEQ ID NO: 158, wherein glutamic acid at position 1 has been changed to aspartic acid.
Aspect E-240. A trivalent polypeptide comprising or consisting essentially of SEQ ID NO: 159, wherein glutamic acid at position 1 has been changed to aspartic acid.
Aspect E-241 A trivalent polypeptide comprising or consisting essentially of SEQ ID NO: 160, wherein glutamic acid at position 1 has been changed to aspartic acid.
Aspect E-242: A trivalent polypeptide comprising or consisting essentially of SEQ ID NO: 161, wherein glutamic acid at position 1 is changed to aspartic acid.
Aspect E-243: In at least one (preferably two, more preferably all three) Nanobodies® forming part of SEQ ID NO: 53, one or more (eg, two, three, 4, 5, 6, 7, 8, or 9, 10, 11, or 12) amino acid residues are Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, A trivalent polypeptide comprising or consisting essentially of SEQ ID NO: 53, which has been mutated selected from Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp, and wherein glutamic acid at position 1 has been changed to aspartic acid.
Aspect E-244: In at least one (preferably two, more preferably all three) Nanobodies® forming part of SEQ ID NO: 53, the amino acid residue is Val5Leu, Ala14Pro, Ser19R, Ile20Leu. Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54D, wherein the glutamic acid at position 1 has been changed to aspartic acid, including or essentially consisting of SEQ ID NO: 53. A trivalent polypeptide.
Aspect E-245: In at least one (preferably two, more preferably all three) Nanobodies® forming part of SEQ ID NO: 53, the amino acid residue is at Ser19R, Ile20Leu, Ala74Ser, Gly78Leu. , Ala83Arg, Asp85Glu, Arg105Gln, and Gly54D, which have undergone one or more (eg, 2, 3, 4, 5, 6, 7, or 8) mutations and is number 1 A trivalent polypeptide comprising or consisting essentially of SEQ ID NO: 53, wherein the glutamic acid of has been changed to aspartic acid.
Aspect E-246: In at least one (preferably two, more preferably all three) Nanobodies® forming part of SEQ ID NO: 53, one or more (eg, two, three, SEQ ID NO: 53, wherein 4 or 5) amino acid residues have undergone a mutation selected from Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54D, wherein glutamic acid at position 1 has been changed to aspartic acid. A trivalent polypeptide comprising or consisting essentially of.
Aspect E-247: In at least one (preferably two, more preferably all three) Nanobodies® forming part of SEQ ID NO: 53, wherein the amino acid residue is Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln. And a trivalent polypeptide comprising or consisting essentially of SEQ ID NO: 53, which has been mutated with Gly54D and wherein the glutamic acid at position 1 has been changed to aspartic acid.
Aspect E-248: In at least one (preferably two, more preferably all three) Nanobodies® forming part of SEQ ID NO: 53, one or more (eg, two, three or SEQ ID NO: 53, wherein 4) amino acid residues have undergone a mutation selected from Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu, wherein glutamic acid at position 1 has been changed to aspartic acid, or An essentially trivalent polypeptide.
Aspect E-249: In at least one (preferably two, more preferably all three) Nanobodies® forming part of SEQ ID NO: 53, one or more (eg, two, three, (4, 5, 6, or 7) amino acid residues have undergone a mutation selected from Ala14Pro, Ser19Arg, Ile20Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu, and glutamic acid at position 1 is aspartic acid. A trivalent polypeptide comprising, or consisting essentially of, SEQ ID NO: 53.
Aspect E-250: In at least one (preferably two, more preferably all three) Nanobodies® forming part of SEQ ID NO: 53, the amino acid residue is:
Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Gly78Leu, Ala83Arg, Asp85Glu, and Arg105Gln,
Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54Asp,
Gly54Asp,
Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Ala83Arg,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Asp85Glu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Arg105Gln,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Asp85Glu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Arg105Gln,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Asp85Glu, and Arg105Gln, or
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, Asp85Glu, and Arg105Gln
A trivalent polypeptide comprising or consisting essentially of SEQ ID NO: 53, wherein the glutamic acid at position 1 has been changed to aspartic acid.
Aspect E-251: Trivalent polypeptide comprising or consisting essentially of SEQ ID NO: 142.
Aspect E-252: Trivalent polypeptide comprising or consisting essentially of SEQ ID NO: 143.
Aspect E-253: Trivalent polypeptide comprising or consisting essentially of SEQ ID NO: 144.
Aspect E-254: Trivalent polypeptide comprising or consisting essentially of SEQ ID NO: 145.
Aspect E-255: Trivalent polypeptide comprising or consisting essentially of one of SEQ ID NOs: 162-165.
Aspect E-256: Dissociation constant (K) of 100 nM to 0.1 nM or less, preferably 10 nM to 0.1 nM or less, more preferably 1 nM to 0.1 nM or less _D The polypeptide according to any one of aspects E-1 to E-255, wherein the polypeptide is capable of binding specifically to protein F of hRSV at
Aspect E-257: 10 ⁴ M ^-1 s ^-1 ~ About 10 ⁷ M ^-1 s ^-1 , Preferably 10 ⁵ M ^-1 s ^-1 -10 ⁷ M ^-1 s ^-1 , More preferably about 10 ⁶ M ^-1 s ^-1 More than k _on The polypeptide according to any one of aspects E-1 to E-256, which is capable of binding specifically to protein F of hRSV at a rate.
Aspect E-258: 10 ^-2 s ^-1 (T _1/2 = 0.69 s) to 10 ^-4 s ^-1 (T _1/2 Is a few days), preferably 10 ^-3 s ^-1 -10 ^-4 s ^-1 , More preferably 5 × 10 ^-3 s ^-1 -10 ^-4 s ^-1 The following k _off The polypeptide according to any one of aspects E-1 to E-257, which is capable of binding specifically to protein F of hRSV at a rate.
Aspect E-259: With an IC50 value of 10 pM to 1000 pM, preferably 10 pM to 250 pM, more preferably 50 pM to 200 pM or less, for example as measured in a microneutralization assay for hRSV Long (eg as described in Example 6). As described above, the polypeptide (polypeptidev) according to any one of aspects E-1 to E-258, which can neutralize hRSV.
Aspect E-260: at least the same, preferably better, at least 10 times better, preferably 20 times better, more preferably 50 times better as compared to the IC50 value obtained with SYNAGIS® With even better IC50 values, even more preferably 60-fold, 70-fold, 80-fold or more than 80-fold, as measured in a microneutralization assay, for example with respect to hRSV Long (as described in example 6). The polypeptide according to any one of aspects E-1 to E-259, which is capable of neutralizing hRSV.
Aspect E-261: Polypeptide according to any one of aspects E-1 to E-260, which is a multispecific construct.
Aspect F-1: One amino acid sequence according to any one of aspects A-1 to A-29, aspects C-1 to C-38, and aspects W-1 to W-38, and / or aspect B -1 to B-18, comprising or consisting essentially of one Nanobody® according to any one of aspects D-1 to D-38 and aspects X-1 to X-38. Monovalent construct.
Aspect F-2: The above amino acid sequence is a domain antibody, an amino acid sequence suitable for use as a domain antibody, a single domain antibody, an amino acid sequence suitable for use as a single domain antibody, “dAb”, as a dAb The monovalent construct according to aspect F-1, wherein the monovalent construct is selected from the group consisting of an amino acid sequence suitable for use, or Nanobody®.
Aspect F-3: Contains one Nanobody (registered trademark) according to any one of Aspects B-1 to B-18, Aspects D-1 to D-38, and Aspects X-1 to X-38 Or a monovalent construct consisting essentially of the same.
Aspect F-4: A monovalent construct selected from the group consisting of SEQ ID NOs: 60-76, 138-141, and 146-157.
Aspect F-5: Use of the monovalent construct according to any one of aspects F-1 to F-4 when preparing a multivalent polypeptide according to any one of aspects E-1 to E-261. use.
Aspect F-6: Use of a monovalent construct according to aspect F-5, wherein the monovalent construct is used as a binding domain or unit when preparing a multivalent construct comprising two or more binding units.
Aspect F-7: Use of a monovalent construct according to aspect F-5 or F-6 in preparing a multivalent polypeptide that preferably exhibits intramolecular binding as compared to intermolecular binding.
Aspect F-8: Multivalent polypeptide exhibits intramolecular binding as compared to intermolecular binding and / or allows multivalent polypeptide to bind simultaneously to all binding sites on protein F of hRSV Wherein the binding domain or the binding unit is linked via a linker, wherein the monovalent construct according to any one of aspects F-5 to F-7 as a binding domain or a binding unit when preparing a multivalent construct. use.
Aspect F-9: Monovalent construct comprises
a) SEQ ID NO: 60 to SEQ ID NO: 76
b) an amino acid sequence having only one amino acid difference from one of SEQ ID NO: 60 to SEQ ID NO: 76, preferably only two, more preferably only one;
i) the amino acid sequence has glutamine (Gln, Q) at position 105 (the position is determined by Kabat numbering);
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
Use of the monovalent construct according to any one of aspects F-5 to F-8, in preparing a multivalent polypeptide selected from the group consisting of:
Aspect F-10: Use of a monovalent construct according to aspect F-9, wherein the monovalent construct consists essentially of one of SEQ ID NO: 60-76.
Aspect F-11: A monovalent construct comprises:
a) SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76
b) only three, preferably only two and more preferably only one amino acid difference from one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75 and SEQ ID NO: 76 An amino acid sequence having
i) the amino acid sequence has glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and / or arginine (Arg, R) at position 83 (the above positions are determined by Kabat numbering; Desired),
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
Use of the monovalent construct according to any one of aspects F-5 to F-8, in preparing a multivalent polypeptide selected from the group consisting of:
Aspect F-12: Monovalent construct comprises
a) SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76, or
b) only three, preferably only two and more preferably only one amino acid difference from one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75 and SEQ ID NO: 76 An amino acid sequence having
i) the amino acid sequence has glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and arginine (Arg, R) at position 83 (the above positions are determined by Kabat numbering) ),
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
Use of the monovalent construct according to embodiment F-11 in preparing a multivalent polypeptide selected from:
Aspect F-13: Aspect F-11 or F, wherein the monovalent consists essentially of one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76 Use of the monovalent construct according to -12.
Aspect F-14: Monovalent construct comprises
a) SEQ ID NO: 65 and SEQ ID NO: 76,
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NO: 65 and SEQ ID NO: 76,
i) the amino acid sequence is aspartic acid (Asp, D) at position 54, glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and / or arginine (Arg, R) at position 83; (The position is determined by Kabat numbering),
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
Use of the monovalent construct according to any one of aspects F-5 to F-8, in preparing a multivalent polypeptide selected from the group consisting of:
Aspect F-15: Monovalent construct comprises
a) SEQ ID NO: 65 and SEQ ID NO: 76,
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NO: 65 and SEQ ID NO: 76,
i) the amino acid sequence has aspartic acid (Asp, D) at position 54, glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and arginine (Arg, R) at position 83; (The above position is obtained by Kabat numbering)
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
Use of the (accordingaspect F-14) monovalent construct according to embodiment F-14 in preparing a multivalent polypeptide selected from:
Aspect F-16: Use of a monovalent construct consisting essentially of SEQ ID NO: 62 in preparing a multivalent polypeptide.
Aspect F-17: Use of a monovalent construct consisting essentially of SEQ ID NO: 65 in preparing a multivalent polypeptide.
Aspect F-18: Use of a monovalent construct consisting essentially of SEQ ID NO: 76 in preparing a multivalent polypeptide.
Aspect F-19: Monovalent construct comprises
a) SEQ ID NO: 146 to SEQ ID NO: 153
b) an amino acid sequence having only one, preferably two, more preferably only one amino acid difference from one of SEQ ID NOs: 146 to 153,
i) the amino acid sequence has proline (Pro, P) at position 14, arginine (Arg, R) at position 19, leucine (Leu, L) at position 20, and leucine (Leu, L) at position 108 (The above position is determined by Kabat numbering),
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
Use of the monovalent construct according to any one of aspects F-5 to F-8, in preparing a multivalent polypeptide selected from the group consisting of:
Aspect F-20: Use of a monovalent construct according to Aspect F-19, wherein the monovalent construct consists essentially of one of SEQ ID NOs: 146-153.
Aspect F-21: A monovalent construct comprises:
a) SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153,
i) the amino acid sequence comprises proline (Pro, P) at position 14, arginine (Arg, R) at position 19, leucine (Leu, L) at position 20, and leucine (Leu, L) at position 108; Arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85, and / or glutamine (Gln, Q) at position 105 (the position is determined by Kabat numbering);
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
Use of the monovalent construct according to any one of aspects F-5 to F-8, in preparing a multivalent polypeptide selected from the group consisting of:
Aspect F-22: Monovalent construct comprises
a) SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153,
i) the amino acid sequence is
SEQ ID NO: 146 (this amino acid sequence preferably has glutamine (Gln, Q) at position 105);
SEQ ID NO: 147 (this amino acid sequence preferably has arginine (Arg, R) at position 83 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 148 (this amino acid sequence preferably has glutamic acid (Glu, E) at position 85 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 149 (this amino acid sequence preferably has arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 151 (this amino acid sequence preferably has arginine (Arg, R) at position 83);
SEQ ID NO: 152 (this amino acid sequence preferably has glutamic acid (Glu, E) at position 85);
SEQ ID NO: 153 (this amino acid sequence preferably has arginine (Arg, R) at position 83 and glutamic acid (Glu, E) at position 85)
The amino acid sequence has a proline (Pro, P) at position 14 and an arginine (Arg, R) at position 19, as in the case of having only 3, preferably 2, and more preferably only 1 amino acid difference. Leucine (Leu, L) at position 20, Leucine (Leu, L) at position 108, arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85, and / or glutamine at position 105 (Gln, Q) (the position is determined by Kabat numbering),
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
Use of the monovalent construct according to embodiment F-21 in preparing a multivalent polypeptide selected from:
Aspect F-23: The monovalent construct according to aspect F-21 or F-22, wherein the monovalent consists essentially of one of SEQ ID NOs: 146-149 and SEQ ID NOs: 151-153. use.
Aspect F-24: Monovalent construct consists essentially of SEQ ID NO: 5 and comprises one or more (eg, 2, 3, 4, 5, 6, 7, 8, 9 or 10, , 11 or 12) amino acids residues undergo Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly-selected mutations from Gly54Ap. Use of the monovalent construct according to any one of aspects F-5 to F-8 in preparing a polypeptide.
Aspect F-25: Monovalent construct consists essentially of SEQ ID NO: 5, wherein the amino acid residues are Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, GlnGLe, Gln108Lu. Use of a monovalent construct according to embodiment F-24 in the preparation of a multivalent polypeptide, which has been subjected to.
Aspect F-26: monovalent construct consists essentially of SEQ ID NO: 5, wherein one or more (eg, 2, 3, 4, 5, 6, 7, or 8) amino acid residues are Use of a monovalent construct according to aspect F-24 in preparing a multivalent polypeptide, mutated selected from Ser19R, Ile20Leu, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54D.
Aspect F-27: monovalent construct consisting essentially of SEQ ID NO: 5, wherein one or more (eg, 2, 3, 4, or 5) amino acid residues is Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Use of a monovalent construct according to embodiment F-24 in preparing a multivalent polypeptide, which has been mutated and selected from Gly54D.
Aspect F-28: An aspect in preparing a multivalent polypeptide wherein the monovalent construct consists essentially of SEQ ID NO: 5 and the amino acid residues have been mutated for Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54D. Use of a monovalent construct according to F-27.
Aspect F-29: monovalent construct consisting essentially of SEQ ID NO: 5, wherein one or more (eg, two, three or four) amino acid residues is selected from Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu. Use of the monovalent construct according to any one of aspects F-5 to F-8, in preparing a multivalent polypeptide having undergone a mutation.
Aspect F-30: Monovalent construct consists essentially of SEQ ID NO: 5, wherein one or more (eg, 2, 3, 4, 5, 6, or 7) amino acid residues are Ala14Pro, The monovalent construct according to any one of aspects F-5 to F-8, for preparing a multivalent polypeptide having a mutation selected from Ser19Arg, Ile20Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu. Use of.
Aspect F-31: wherein the monovalent construct consists essentially of SEQ ID NO: 5, wherein the amino acid residue is
Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Gly78Leu, Ala83Arg, Asp85Glu, and Arg105Gln,
Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54Asp,
Gly54Asp,
Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Ala83Arg,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Asp85Glu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Arg105Gln,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Asp85Glu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Arg105Gln,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Asp85Glu, and Arg105Gln, or
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, Asp85Glu, and Arg105Gln
Use of the monovalent construct according to embodiment F-24 in preparing a multivalent polypeptide, which has been mutated.
Aspect F-32: wherein the monovalent construct is
a) SEQ ID NO: 138 to SEQ ID NO: 141 and SEQ ID NO: 154 to SEQ ID NO: 157;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 138 to 141 and 154 to 157,
i) the amino acid sequence has aspartic acid (Gln, Q) at position 1 (the position is determined by Kabat numbering);
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
Use of the monovalent construct according to any one of aspects F-5 to F-8, in preparing a multivalent polypeptide selected from the group consisting of:
Aspect F-33: Use of a monovalent construct according to aspect F-32, wherein the monovalent construct consists essentially of one of SEQ ID NOs: 138-141 and SEQ ID NOs: 154-157.
Aspect F-34: Any of aspects F-5 to F-8 for preparing a multivalent polypeptide, wherein the monovalent construct consists essentially of SEQ ID NO: 5 and the glutamic acid at position 1 has been changed to aspartic acid. Use of a monovalent construct according to any one of the preceding claims.
Aspect F-35: Any of aspects F-5 to F-8 for preparing a multivalent polypeptide, wherein the monovalent construct consists essentially of SEQ ID NO: 62 and the glutamic acid at position 1 has been changed to aspartic acid. Use of a monovalent construct according to any one of the preceding claims.
Aspect F-36: Any of aspects F-5 to F-8 for preparing a multivalent polypeptide, wherein the monovalent construct consists essentially of SEQ ID NO: 65 and the glutamic acid at position 1 has been changed to aspartic acid. Use of a monovalent construct according to any one of the preceding claims.
Aspect F-37: Any of aspects F-5 to F-8 for preparing a multivalent polypeptide, wherein the monovalent construct consists essentially of SEQ ID NO: 76 and the glutamic acid at position 1 has been changed to aspartic acid. Use of a monovalent construct according to any one of the preceding claims.
Aspect F-38: Any of aspects F-5 to F-8 for preparing a multivalent polypeptide, wherein the monovalent construct consists essentially of SEQ ID NO: 75 and the glutamic acid at position 1 has been changed to aspartic acid. Use of a monovalent construct according to any one of the preceding claims.
Aspect F-39: Any of aspects F-5 to F-8 for preparing a multivalent polypeptide, wherein the monovalent construct consists essentially of SEQ ID NO: 147 and the glutamic acid at position 1 has been changed to aspartic acid. Use of a monovalent construct according to any one of the preceding claims.
Aspect F-40: Any of aspects F-5 to F-8 for preparing a multivalent polypeptide, wherein the monovalent construct consists essentially of SEQ ID NO: 149 and glutamic acid at position 1 has been changed to aspartic acid. Use of a monovalent construct according to any one of the preceding claims.
Aspect F-41: Any of aspects F-5 to F-8 for preparing a multivalent polypeptide, wherein the monovalent construct consists essentially of SEQ ID NO: 153 and the glutamic acid at position 1 has been changed to aspartic acid. Use of a monovalent construct according to any one of the preceding claims.
Aspect F-42: monovalent construct consists essentially of SEQ ID NO: 5 and comprises one or more (eg, 2, 3, 4, 5, 6, 7, 8, 9 or 10, , 11 or 12) amino acids residues are mutated from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly selected from Gly54Ap. Use of the monovalent construct according to any one of aspects F-5 to F-8, in the preparation of a multivalent polypeptide, wherein the glutamic acid at the position is changed to aspartic acid.
Aspect F-43: monovalent construct consisting essentially of SEQ ID NO: 5, wherein the amino acid residues are Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, GlnGLe, and Gln108Ly. Use of the monovalent construct according to aspect F-42, in preparing a multivalent polypeptide, wherein the glutamic acid at position 1 has been changed to aspartic acid.
Aspect F-44: monovalent construct consists essentially of SEQ ID NO: 5, wherein one or more (eg, 2, 3, 4, 5, 6, 7, or 8) amino acid residues are , Ser19R, Ile20Leu, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and a mutation selected from Gly54D, wherein the glutamic acid at position 1 has been changed to aspartic acid. Use of a monovalent construct according to aspect F-42.
Aspect F-45: monovalent construct consists essentially of SEQ ID NO: 5, wherein one or more (eg, 2, 3, 4, or 5) amino acid residues is Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Use of the monovalent construct according to embodiment F-42, in preparing a multivalent polypeptide, wherein the monovalent construct has undergone a mutation selected from Gly54D and wherein the glutamic acid at position 1 has been changed to aspartic acid.
Aspect F-46: monovalent construct consisting essentially of SEQ ID NO: 5, wherein the amino acid residue has been mutated for Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54D, changing glutamic acid at position 1 to aspartic acid Use of the monovalent construct according to embodiment F-42, in preparing a multivalent polypeptide.
Aspect F-47: monovalent construct consists essentially of SEQ ID NO: 5, wherein one or more (eg, two, three or four) amino acid residues is selected from Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu. The monovalent construct according to any one of aspects F-5 to F-8, when preparing a multivalent polypeptide, wherein the monovalent construct has undergone a mutation and glutamic acid at position 1 has been changed to aspartic acid. use.
Aspect F-48: monovalent construct consists essentially of SEQ ID NO: 5, and comprises one or more (eg, 2, 3, 4, 5, 6, 7, 8, or 9, 10, , 11 or 12) have undergone a mutation selected from Ala14Pro, Ser19Arg, Ile20Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu, and the glutamic acid at position 1 has been changed to aspartic acid. Use of the monovalent construct according to any one of aspects F-5 to F-8 in preparing a multivalent polypeptide.
Aspect F-49: monovalent construct consisting essentially of SEQ ID NO: 5, wherein the amino acid residue is
Glu1Asp,
Glu1Asp, Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Glu1Asp, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Glu1Asp, Gly78Leu, Ala83Arg, Asp85Glu, and Arg105Gln;
Glu1Asp, Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Glu1Asp, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Glu1Asp, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54Asp,
Glu1Asp and Gly54Asp,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Ala83Arg;
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Asp85Glu,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Arg105Gln,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Asp85Glu,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Arg105Gln, Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Asp85Glu, and Arg105, Arg
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, Asp85Glu, and Arg105Gln
Use of a monovalent construct according to embodiment F-42 in preparing a multivalent construct, which has been mutated.
Aspect F-50: Use of two monovalent constructs according to any one of aspects F-1 to F-4 in preparing a bivalent polypeptide.
Aspect F-51: Use of two monovalent constructs according to aspect F-50 in preparing a bivalent polypeptide that preferably exhibits intramolecular binding as compared to intermolecular binding.
Aspect F-52: Bivalent polypeptide exhibits intramolecular binding as compared to intermolecular binding, and / or allows the bivalent polypeptide to bind simultaneously to both binding sites on protein F of hRSV. Use of two monovalent constructs according to aspect F-50 or F-51, as a binding domain or binding unit, in preparing a bivalent polypeptide, wherein the binding domain or binding unit is linked via a linker.
Aspect F-53: wherein the monovalent construct is
a) SEQ ID NO: 60 to SEQ ID NO: 76
b) an amino acid sequence having only one amino acid difference from one of SEQ ID NO: 60 to SEQ ID NO: 76, preferably only two, more preferably only one;
i) the amino acid sequence has glutamine (Gln, Q) at position 105 (the position is determined by Kabat numbering);
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
Use of two monovalent constructs according to any one of aspects F-50 to F-52 in preparing a bivalent polypeptide, selected from:
Aspect F-54: Use of two monovalent constructs according to aspect F-53, wherein the two monovalent constructs are identical.
Aspect F-55: Use of two monovalent constructs according to aspect F-53 or F-54, wherein the two monovalent constructs consist essentially of one of SEQ ID NO: 60-76.
Aspect F-56: wherein the monovalent construct comprises:
a) SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76
b) only three, preferably only two and more preferably only one amino acid difference from one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75 and SEQ ID NO: 76 An amino acid sequence having
i) the amino acid sequence has glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and / or arginine (Arg, R) at position 83 (the above positions are determined by Kabat numbering; Desired),
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
Use of two monovalent constructs according to any one of aspects F-50 to F-52 in preparing a bivalent polypeptide, selected from:
Aspect F-57: wherein the monovalent construct is
a) SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76
b) only three, preferably only two and more preferably only one amino acid difference from one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75 and SEQ ID NO: 76 An amino acid sequence having
i) the amino acid sequence has glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and arginine (Arg, R) at position 83 (the above positions are determined by Kabat numbering) ),
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
Use of two monovalent constructs according to embodiment F-56 in preparing a bivalent polypeptide selected from:
Aspect F-58: Use of two monovalent constructs according to aspect F-56 or F-57, wherein the two monovalent constructs are the same.
Aspect F-59: Aspect F-58, wherein the two monovalent constructs consist essentially of one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76. Use of two monovalent constructs as described in 1.
Aspect F-60: Monovalent construct comprises
a) SEQ ID NO: 65 and SEQ ID NO: 76,
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NO: 65 and SEQ ID NO: 76,
i) the amino acid sequence is aspartic acid (Asp, D) at position 54, glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and / or arginine (Arg, R) at position 83; (The position is determined by Kabat numbering),
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
Use of two monovalent constructs according to any one of aspects F-50 to F-52 in preparing a bivalent polypeptide, selected from:
Aspect F-61: wherein the monovalent construct is
a) SEQ ID NO: 65 and SEQ ID NO: 76,
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NO: 65 and SEQ ID NO: 76,
i) the amino acid sequence has aspartic acid (Asp, D) at position 54, glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and arginine (Arg, R) at position 83; (The above position is obtained by Kabat numbering)
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Use of two monovalent constructs according to embodiment F-60 in the preparation of a bivalent polypeptide, selected from an amino acid sequence (as defined herein).
Aspect F-62: Use of two monovalent constructs according to aspect F-60 or F-61, wherein the two monovalent constructs are the same.
Aspect F-63: Use of two monovalent constructs consisting essentially of SEQ ID NO: 62 in preparing a bivalent polypeptide.
Aspect F-64: Use of two monovalent constructs consisting essentially of SEQ ID NO: 65 in preparing a bivalent polypeptide.
Aspect F-65: Use of two monovalent constructs consisting essentially of SEQ ID NO: 76 in preparing a bivalent polypeptide.
Aspect F-66: wherein the monovalent construct comprises:
a) SEQ ID NO: 146 to SEQ ID NO: 153
b) an amino acid sequence having only one, preferably two, more preferably only one amino acid difference from one of SEQ ID NOs: 146 to 153,
i) the amino acid sequence has proline (Pro, P) at position 14, arginine (Arg, R) at position 19, leucine (Leu, L) at position 20, and leucine (Leu, L) at position 108 (The above position is determined by Kabat numbering),
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
Use of two monovalent constructs according to any one of aspects F-50 to F-52 in preparing a bivalent polypeptide, selected from:
Aspect F-67: Use of two monovalent constructs according to aspect F-66, wherein the two monovalent constructs are identical.
Aspect F-68: Use of a two monovalent construct according to aspect F-66 or F-67, wherein the two monovalent constructs consist essentially of one of SEQ ID NOs: 146-153.
Aspect F-69: monovalent construct comprises
a) SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153,
i) the amino acid sequence comprises proline (Pro, P) at position 14, arginine (Arg, R) at position 19, leucine (Leu, L) at position 20, and leucine (Leu, L) at position 108; Arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85, and / or glutamine (Gln, Q) at position 105 (the position is determined by Kabat numbering);
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
Use of two monovalent constructs according to any one of aspects F-50 to F-52 in preparing a bivalent polypeptide, selected from:
Aspect F-70: monovalent construct comprises
a) SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153,
i) the amino acid sequence is
SEQ ID NO: 146 (this amino acid sequence preferably has glutamine (Gln, Q) at position 105);
SEQ ID NO: 147 (this amino acid sequence preferably has arginine (Arg, R) at position 83 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 148 (this amino acid sequence preferably has glutamic acid (Glu, E) at position 85 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 149 (this amino acid sequence preferably has arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 151 (this amino acid sequence preferably has arginine (Arg, R) at position 83);
SEQ ID NO: 152 (this amino acid sequence preferably has glutamic acid (Glu, E) at position 85);
SEQ ID NO: 153 (this amino acid sequence preferably has arginine (Arg, R) at position 83 and glutamic acid (Glu, E) at position 85)
The amino acid sequence has a proline (Pro, P) at position 14 and an arginine (Arg, R) at position 19, as in the case of having only 3, preferably 2, and more preferably only 1 amino acid difference. Leucine (Leu, L) at position 20, Leucine (Leu, L) at position 108, arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85, and / or glutamine at position 105 (Gln, Q) (the position is determined by Kabat numbering),
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
Use of two monovalent constructs according to embodiment F-69 in preparing a bivalent polypeptide selected from:
Aspect F-71: Use of two monovalent constructs according to aspect F-69 or F-70, wherein the two monovalent constructs are the same.
Aspect F-72: of two monovalent constructs according to aspect F-71, wherein the two monovalent constructs consist essentially of one of SEQ ID NOs: 146-149 and SEQ ID NOs: 151-153. use.
Aspect F-73: monovalent construct consists essentially of SEQ ID NO: 5, and comprises one or more (eg, 2, 3, 4, 5, 6, 7, 8, 9 or 10, , 11 or 12) amino acid residues undergo Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Ap mutated from Gly54Ap. Use of two monovalent constructs according to any one of aspects F-50 to F-52 in preparing a polypeptide.
Aspect F-74: monovalent construct consisting essentially of SEQ ID NO: 5, wherein the amino acid residues are Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, GlnGle54, GlnGLe. Use of two monovalent constructs according to embodiment F-73 in preparing a bivalent polypeptide, which has received
Aspect F-75: monovalent construct consists essentially of SEQ ID NO: 5, wherein one or more (eg, 2, 3, 4, 5, 6, 7, or 8) amino acid residues are Use of two monovalent constructs according to embodiment F-73, in preparing a divalent polypeptide, mutated from Ser19R, Ile20Leu, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54D. .
Aspect F-76: monovalent construct consisting essentially of SEQ ID NO: 5, wherein one or more (eg, 2, 3, 4, or 5) amino acid residues is Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Use of two monovalent constructs according to embodiment F-73 in preparing a divalent polypeptide, which has been mutated selected from Gly54D.
Aspect F-77: An aspect in preparing a bivalent polypeptide wherein the monovalent construct consists essentially of SEQ ID NO: 5 and the amino acid residues have been mutated Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54D. Use of two monovalent constructs according to F-76.
Aspect F-78: One or more (eg, two, three, or four) amino acid residues are selected from Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu, wherein the monovalent construct consists essentially of SEQ ID NO: 5. Use of two monovalent constructs according to any one of aspects F-50 to F-52 in preparing a bivalent polypeptide, which has been mutated.
Aspect F-79: monovalent construct consisting essentially of SEQ ID NO: 5, wherein one or more (eg, 2, 3, 4, 5, 6, or 7) amino acid residues is Ala14Pro, Two of any one of embodiments F-50 to F-52 for preparing a bivalent polypeptide, which has been mutated selected from Ser19Arg, Ile20Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu. Use of Valent Constructs.
Aspect F-80: monovalent construct consists essentially of SEQ ID NO: 5, wherein the amino acid residue is
Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Gly78Leu, Ala83Arg, Asp85Glu, and Arg105Gln,
Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54Asp,
Gly54Asp,
Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Ala83Arg,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Asp85Glu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Arg105Gln,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Asp85Glu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Arg105Gln,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Asp85Glu, and Arg105Gln, or
Use of two monovalent constructs according to embodiment F-73 in preparing a bivalent polypeptide, mutated Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, Asp85Glu, and Arg105Gln.
Aspect F-81: wherein the monovalent construct is
a) SEQ ID NO: 138 to SEQ ID NO: 141 and SEQ ID NO: 154 to SEQ ID NO: 157;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 138 to 141 and 154 to 157,
i) the amino acid sequence has aspartic acid (Asp, D) at position 1 (the position is determined by Kabat numbering);
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
Use of the monovalent construct according to any one of aspects F-50 to F-52, in preparing a bivalent polypeptide selected from the group consisting of:
Aspect F-82: Use of a monovalent construct according to aspect F-81, wherein the monovalent construct consists essentially of one of SEQ ID NOs: 138-141 and SEQ ID NOs: 154-157.
Aspect F-83: Any of aspects F-50 to F-52 for preparing a divalent polypeptide, wherein the monovalent construct consists essentially of SEQ ID NO: 5 and the glutamic acid at position 1 has been changed to aspartic acid. Use of a monovalent construct according to any one of the preceding claims.
Aspect F-84: Any of aspects F-50 to F-52 for preparing a divalent polypeptide, wherein the monovalent construct consists essentially of SEQ ID NO: 62 and the glutamic acid at position 1 has been changed to aspartic acid. Use of a monovalent construct according to any one of the preceding claims.
Aspect F-85: Any of aspects F-50 to F-52 for preparing a divalent polypeptide, wherein the monovalent construct consists essentially of SEQ ID NO: 65 and the glutamic acid at position 1 has been changed to aspartic acid. Use of a monovalent construct according to any one of the preceding claims.
Aspect F-86: Any of aspects F-50 to F-52 for preparing a bivalent polypeptide, wherein the monovalent construct consists essentially of SEQ ID NO: 76 and the glutamic acid at position 1 has been changed to aspartic acid. Use of a monovalent construct according to any one of the preceding claims.
Aspect F-87: Any of aspects F-50 to F-52 for preparing a bivalent polypeptide, wherein the monovalent construct consists essentially of SEQ ID NO: 75 and the glutamic acid at position 1 has been changed to aspartic acid. Use of a monovalent construct according to any one of the preceding claims.
Aspect F-88: Any of aspects F-50 to F-52 for preparing a bivalent polypeptide, wherein the monovalent construct consists essentially of SEQ ID NO: 147 and the glutamic acid at position 1 has been changed to aspartic acid. Use of a monovalent construct according to any one of the preceding claims.
Aspect F-89: Any of aspects F-50 to F-52 for preparing a divalent polypeptide, wherein the monovalent construct consists essentially of SEQ ID NO: 149 and glutamic acid at position 1 has been changed to aspartic acid. Use of a monovalent construct according to any one of the preceding claims.
Aspect F-90: Any of aspects F-50 to F-52 for preparing a divalent polypeptide, wherein the monovalent construct consists essentially of SEQ ID NO: 153 and the glutamic acid at position 1 has been changed to aspartic acid. Use of a monovalent construct according to any one of the preceding claims.
Aspect F-91: monovalent construct consists essentially of SEQ ID NO: 5, and comprises one or more (eg, 2, 3, 4, 5, 6, 7, 8, 9 or 10, , 11 or 12) amino acids residues undergo a Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly selection from a Gly54Ap mutation. Use of the monovalent construct according to any one of aspects F-50 to F-52, in the preparation of a bivalent polypeptide, wherein the glutamic acid in position is changed to aspartic acid.
Aspect F-92: monovalent construct consisting essentially of SEQ ID NO: 5, wherein the amino acid residues are Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, GlnGle, GlnGle54. Use of the monovalent construct according to embodiment F-91, in preparing a divalent polypeptide, wherein the glutamic acid at position 1 has been changed to aspartic acid.
Aspect F-93: monovalent construct consists essentially of SEQ ID NO: 5, wherein one or more (eg, 2, 3, 4, 5, 6, 7, or 8) amino acid residues are , Ser19R, Ile20Leu, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and a mutation selected from Gly54D, wherein the glutamic acid at position 1 has been changed to aspartic acid. Use of a monovalent construct according to embodiment F-91.
Aspect F-94: monovalent construct consists essentially of SEQ ID NO: 5, wherein one or more (eg, 2, 3, 4, or 5) amino acid residues is Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, And a mutation selected from Gly54D and wherein the glutamic acid at position 1 has been changed to aspartic acid, using the monovalent construct according to embodiment F-91, in preparing a bivalent polypeptide.
Aspect F-95: monovalent construct consisting essentially of SEQ ID NO: 5, wherein the amino acid residue has been mutated for Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54D, changing glutamic acid at position 1 to aspartic acid Use of the monovalent construct according to embodiment F-94, in preparing a bivalent polypeptide.
Aspect F-96: monovalent construct consists essentially of SEQ ID NO: 5, wherein one or more (eg, two, three or four) amino acid residues is selected from Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu. The monovalent construct according to any one of aspects F-50 to F-52, when preparing a divalent polypeptide, wherein the monovalent construct has undergone a mutation and the glutamic acid at position 1 has been changed to aspartic acid. use.
Aspect F-97: monovalent construct consisting essentially of SEQ ID NO: 5, wherein one or more (eg, 2, 3, 4, 5, 6, or 7) amino acid residues is Ala14Pro, Aspect F-50 of preparing a divalent polypeptide having a mutation selected from Ser19Arg, Ile20Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu, wherein glutamic acid at position 1 has been changed to aspartic acid. Use of a monovalent construct according to any one of F-52.
Aspect F-98: monovalent construct consists essentially of SEQ ID NO: 5, wherein the amino acid residue is
Glu1Asp,
Glu1Asp, Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Glu1Asp, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Glu1Asp, Gly78Leu, Ala83Arg, Asp85Glu, and Arg105Gln;
Glu1Asp, Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Glu1Asp, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Glu1Asp, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54Asp,
Glu1Asp and Gly54Asp, Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Ala83Arg;
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Asp85Glu,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Arg105Gln,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Asp85Glu,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Arg105Gln,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Asp85Glu, and Arg105Gln, or
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, Asp85Glu, and Arg105Gln
Use of a monovalent construct according to embodiment F-91 in preparing a bivalent construct, which has been mutated.
Aspect F-99: Use of a three monovalent construct according to any one of aspects F-1 to F-4 in preparing a trivalent polypeptide.
Aspect F-100: Use of a three monovalent construct according to aspect F-99 in preparing a trivalent polypeptide that preferably exhibits intramolecular binding as compared to intermolecular binding.
Aspect F-101: Trivalent polypeptide exhibits intramolecular binding as compared to intermolecular binding, and / or trivalent polypeptide can be simultaneously bound to all three binding sites on protein F of hRSV As such, the three monovalent constructs according to aspect F-99 or F-100 as binding domains or binding units in preparing a trivalent polypeptide, wherein the binding domains or binding units are linked via a linker. use.
Aspect F-102: wherein the monovalent construct comprises:
a) SEQ ID NO: 60 to SEQ ID NO: 76
b) an amino acid sequence having only one amino acid difference from one of SEQ ID NO: 60 to SEQ ID NO: 76, preferably only two, more preferably only one;
i) the amino acid sequence has glutamine (Gln, Q) at position 105 (the position is determined by Kabat numbering);
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
Use of three monovalent constructs according to any one of aspects F-99 to F-101 in preparing a trivalent polypeptide selected from:
Aspect F-103: Use of three monovalent constructs according to aspect F-102, wherein the three monovalent constructs are identical.
Aspect F-104: Use of a three monovalent construct according to aspect F-102 or F-103, wherein the three monovalent constructs consist essentially of one of SEQ ID NO: 60-76.
Aspect F-105: wherein the monovalent construct comprises:
a) SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76
b) only three, preferably only two and more preferably only one amino acid difference from one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75 and SEQ ID NO: 76 An amino acid sequence having
i) the amino acid sequence has glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and / or arginine (Arg, R) at position 83 (the above positions are determined by Kabat numbering; Desired),
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
Use of three monovalent constructs according to any one of aspects F-99 to F-101 in preparing a trivalent polypeptide selected from:
Aspect F-106: A monovalent construct has
a) SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76
b) only three, preferably only two and more preferably only one amino acid difference from one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75 and SEQ ID NO: 76 An amino acid sequence having
i) the amino acid sequence has glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and arginine (Arg, R) at position 83 (the above positions are determined by Kabat numbering) ),
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
Use of the three monovalent constructs of embodiment F-105 in preparing a trivalent polypeptide selected from:
Aspect F-107: Use of three monovalent constructs according to aspect F-105 or F-106, wherein the three monovalent constructs are identical.
Aspect F-108: Aspect F-107, wherein the three monovalent constructs consist essentially of one of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 75, and SEQ ID NO: 76. Use of three monovalent constructs as described in 1.
Aspect F-109: wherein the monovalent construct comprises:
a) SEQ ID NO: 65 and SEQ ID NO: 76,
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NO: 65 and SEQ ID NO: 76,
i) the amino acid sequence is aspartic acid (Asp, D) at position 54, glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and / or arginine (Arg, R) at position 83; (The position is determined by Kabat numbering),
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
Use of three monovalent constructs according to any one of aspects F-99 to F-101 in preparing a trivalent polypeptide selected from:
Aspect F-110: A monovalent construct comprises: a) SEQ ID NO: 65 and SEQ ID NO: 76;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NO: 65 and SEQ ID NO: 76,
i) the amino acid sequence has aspartic acid (Asp, D) at position 54, glutamine (Gln, Q) at position 105, leucine (Leu, L) at position 78, and arginine (Arg, R) at position 83; (The above position is obtained by Kabat numbering)
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
Use of the three monovalent constructs according to embodiment F-109 in preparing a trivalent polypeptide selected from:
Aspect F-111: Use of three monovalent constructs according to aspect F-109 or F-110, wherein the three monovalent constructs are identical.
Aspect F-112: Use of three monovalent constructs consisting essentially of SEQ ID NO: 62 in preparing a trivalent polypeptide.
Aspect F-113: Use of three monovalent constructs consisting essentially of SEQ ID NO: 65 in preparing a trivalent polypeptide.
Aspect F-114: Use of three monovalent constructs consisting essentially of SEQ ID NO: 76 in preparing a trivalent polypeptide.
Aspect F-115: wherein the monovalent construct comprises:
a) SEQ ID NO: 146 to SEQ ID NO: 153
b) an amino acid sequence having only one, preferably two, more preferably only one amino acid difference from one of SEQ ID NOs: 146 to 153,
i) the amino acid sequence has proline (Pro, P) at position 14, arginine (Arg, R) at position 19, leucine (Leu, L) at position 20, and leucine (Leu, L) at position 108 (The above position is determined by Kabat numbering),
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
Use of three monovalent constructs according to any one of aspects F-99 to F-101 in preparing a trivalent polypeptide selected from:
Aspect F-116: Use of three monovalent constructs according to aspect F-115, wherein the three monovalent constructs are identical.
Aspect F-117: Use of a three monovalent construct according to aspect F-115 or F-116, wherein the three monovalent constructs consist essentially of one of SEQ ID NOs: 146-153.
Aspect F-118: wherein the monovalent construct comprises:
a) SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153,
i) the amino acid sequence comprises proline (Pro, P) at position 14, arginine (Arg, R) at position 19, leucine (Leu, L) at position 20, and leucine (Leu, L) at position 108; Arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85, and / or glutamine (Gln, Q) at position 105 (the position is determined by Kabat numbering);
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
Use of three monovalent constructs according to any one of aspects F-99 to F-101 in preparing a trivalent polypeptide selected from:
Aspect F-119: monovalent construct comprises
a) SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 146 to 149 and SEQ ID NOs: 151 to 153,
i) the amino acid sequence is
SEQ ID NO: 146 (this amino acid sequence preferably has glutamine (Gln, Q) at position 105);
SEQ ID NO: 147 (this amino acid sequence preferably has arginine (Arg, R) at position 83 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 148 (this amino acid sequence preferably has glutamic acid (Glu, E) at position 85 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 149 (this amino acid sequence preferably has arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85 and glutamine (Gln, Q) at position 105);
SEQ ID NO: 151 (this amino acid sequence preferably has arginine (Arg, R) at position 83);
SEQ ID NO: 152 (this amino acid sequence preferably has glutamic acid (Glu, E) at position 85);
SEQ ID NO: 153 (this amino acid sequence preferably has arginine (Arg, R) at position 83 and glutamic acid (Glu, E) at position 85)
The amino acid sequence has a proline (Pro, P) at position 14 and an arginine (Arg, R) at position 19, as in the case of having only 3, preferably 2, and more preferably only 1 amino acid difference. Leucine (Leu, L) at position 20, Leucine (Leu, L) at position 108, arginine (Arg, R) at position 83, glutamic acid (Glu, E) at position 85, and / or glutamine at position 105 (Gln, Q) (the position is determined by Kabat numbering),
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
Use of the three monovalent constructs of embodiment F-118 in preparing a trivalent polypeptide selected from:
Aspect F-120: Use of three monovalent constructs according to aspect F-118 or F-119, wherein the three monovalent constructs are identical.
Aspect F-121: of three monovalent constructs according to aspect F-120, wherein the three monovalent constructs consist essentially of one of SEQ ID NOs: 146-149 and SEQ ID NOs: 151-153. use.
Aspect F-122: monovalent construct consists essentially of SEQ ID NO: 5, and comprises one or more (eg, 2, 3, 4, 5, 6, 7, 8, 9 or 10, 10) , 11 or 12) amino acid residues undergo Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly-selected mutations from Gly54Ap. Use of three monovalent constructs according to any one of aspects F-99 to F-101 in preparing a polypeptide.
Aspect F-123: monovalent construct consisting essentially of SEQ ID NO: 5, wherein the amino acid residues are Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, GlnGLyu, Gln108Lu. Use of the three monovalent constructs according to embodiment F-122 in preparing a trivalent polypeptide, which has been subjected to.
Aspect F-124: wherein the monovalent construct consists essentially of SEQ ID NO: 5, and the amino acid residue is one or more (eg, 2) selected from Ser19R, Ile20Leu, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54D. Use of three monovalent constructs according to embodiment F-122 in preparing a trivalent polypeptide, which has been mutated (1, 3, 4, 5, 6, 7, or 8) .
Aspect F-125: wherein the monovalent construct consists essentially of SEQ ID NO: 5, and the amino acid residue is one or more (eg, 2, 3, 4) selected from Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54D. Use of three monovalent constructs according to embodiment F-122, in preparing a trivalent polypeptide, which has been mutated.
Aspect F-126: Aspect for preparing a trivalent polypeptide wherein the monovalent construct consists essentially of SEQ ID NO: 5 and the amino acid residues have been mutated for Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54D. Use of three monovalent constructs according to F-125.
Aspect F-127: one or more (eg, two, three or four) amino acid residues are selected from Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu, wherein the monovalent construct consists essentially of SEQ ID NO: 5. Use of three monovalent constructs according to any one of aspects F-99 to F-101 in preparing a trivalent polypeptide, which has been mutated.
Aspect F-128: wherein the monovalent construct consists essentially of SEQ ID NO: 5, wherein the amino acid residue is one or more (eg, two, selected from Ala14Pro, Ser19Arg, Ile20Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu). Three, four, five, six or seven) mutated trivalent polypeptides according to any one of aspects F-99 to F-101 in preparing a trivalent polypeptide. Use of Valent Constructs.
Aspect F-129: monovalent construct consisting essentially of SEQ ID NO: 5, wherein the amino acid residue is
Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Gly78Leu, Ala83Arg, Asp85Glu, and Arg105Gln,
Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54Asp,
Gly54Asp,
Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Ala83Arg,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Asp85Glu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Arg105Gln,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Asp85Glu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Arg105Gln,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Asp85Glu, and Arg105Gln, or
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, Asp85Glu, and Arg105Gln
Use of three monovalent constructs according to embodiment F-122 in preparing a trivalent polypeptide, which has been mutated.
Aspect F-130: For preparing a trivalent polypeptide having SEQ ID NO: 77, wherein the amino acid sequence having SEQ ID NO: 62 is linked to at least two additional amino acid sequences having SEQ ID NO: 62 via a 15GS linker. Use of the amino acid sequence having SEQ ID NO: 62.
Aspect F-131: For preparing a trivalent polypeptide having SEQ ID NO: 78, wherein the amino acid sequence having SEQ ID NO: 65 is linked to at least two additional amino acid sequences having SEQ ID NO: 65 via a 15GS linker. Use of the amino acid sequence having SEQ ID NO: 65.
Aspect F-132: In preparing a trivalent polypeptide having SEQ ID NO: 79, wherein the amino acid sequence having SEQ ID NO: 76 is linked to at least two additional amino acid sequences having SEQ ID NO: 76 via a 15GS linker. Use of the amino acid sequence having SEQ ID NO: 76.
Aspect F-133: In preparing a trivalent polypeptide having SEQ ID NO: 158, wherein the amino acid sequence having SEQ ID NO: 75 is linked to at least two additional amino acid sequences having SEQ ID NO: 75 via a 15GS linker. Use of the amino acid sequence having SEQ ID NO: 75.
Aspect F-134: In preparing a trivalent polypeptide having SEQ ID NO: 159, wherein the amino acid sequence having SEQ ID NO: 147 is linked to at least two additional amino acid sequences having SEQ ID NO: 147 via a 15GS linker. Use of the amino acid sequence having SEQ ID NO: 147.
Aspect F-135: In preparing a trivalent polypeptide having SEQ ID NO: 160, wherein the amino acid sequence having SEQ ID NO: 149 is linked to at least two additional amino acid sequences having SEQ ID NO: 149 via a 15GS linker. Use of the amino acid sequence having SEQ ID NO: 149.
Aspect F-136: In preparing a trivalent polypeptide having SEQ ID NO: 161, wherein the amino acid sequence having SEQ ID NO: 153 is linked to at least two additional amino acid sequences having SEQ ID NO: 153 via a 15GS linker. Use of the amino acid sequence having SEQ ID NO: 153.
Aspect F-137: wherein the monovalent construct comprises:
a) SEQ ID NO: 138 to SEQ ID NO: 141 and SEQ ID NO: 154 to SEQ ID NO: 157;
b) an amino acid sequence having only 3, preferably 2, and more preferably only 1 amino acid difference from one of SEQ ID NOs: 138 to 141 and 154 to 157,
i) the amino acid sequence has aspartic acid (Asp, D) at position 1 (the position is determined by Kabat numbering);
ii) the amino acid sequence has the same, nearly the same, or a higher affinity as compared to an amino acid sequence having no three, two or one amino acid difference (as said affinity is measured by surface plasmon resonance); That bind to protein F of hRSV and / or wherein said amino acid sequence has the same, approximately the same, or higher potency (as compared to an amino acid sequence without three, two or one amino acid difference) Amino acid sequence (as defined herein)
Use of the monovalent construct according to any one of aspects F-99 to F-101 in preparing a trivalent polypeptide selected from:
Aspect F-138: Use of a monovalent construct according to aspect F-137, wherein the monovalent construct consists essentially of one of SEQ ID NOs: 138-141 and SEQ ID NOs: 154-157.
Aspect F-139: Any of aspects F-99 to F-101 for preparing a trivalent polypeptide, wherein the monovalent construct consists essentially of SEQ ID NO: 5 and the glutamic acid at position 1 has been changed to aspartic acid. Use of a monovalent construct according to any one of the preceding claims.
Aspect F-140: Any of aspects F-99 to F-101 in preparing a trivalent polypeptide, wherein the monovalent construct consists essentially of SEQ ID NO: 62 and the glutamic acid at position 1 has been changed to aspartic acid. Use of a monovalent construct according to any one of the preceding claims.
Aspect F-141: Any of aspects F-99 to F-101 for preparing a trivalent polypeptide, wherein the monovalent construct consists essentially of SEQ ID NO: 65 and the glutamic acid at position 1 has been changed to aspartic acid. Use of a monovalent construct according to any one of the preceding claims.
Aspect F-142: Any of aspects F-99 to F-101 in preparing a trivalent polypeptide, wherein the monovalent construct consists essentially of SEQ ID NO: 76 and the glutamic acid at position 1 has been changed to aspartic acid. Use of a monovalent construct according to any one of the preceding claims.
Aspect F-143: Any of aspects F-99 to F-101 for preparing a trivalent polypeptide, wherein the monovalent construct consists essentially of SEQ ID NO: 75 and the glutamic acid at position 1 has been changed to aspartic acid. Use of a monovalent construct according to any one of the preceding claims.
Aspect F-144: Any of aspects F-99 to F-101 for preparing a trivalent polypeptide, wherein the monovalent construct consists essentially of SEQ ID NO: 147 and the glutamic acid at position 1 has been changed to aspartic acid. Use of a monovalent construct according to any one of the preceding claims.
Aspect F-145: Any of aspects F-99 to F-101 for preparing a trivalent polypeptide, wherein the monovalent construct consists essentially of SEQ ID NO: 149 and the glutamic acid at position 1 has been changed to aspartic acid. Use of a monovalent construct according to any one of the preceding claims.
Aspect F-146: Any of aspects F-99 to F-101 for preparing a trivalent polypeptide, wherein the monovalent construct consists essentially of SEQ ID NO: 153 and the glutamic acid at position 1 has been changed to aspartic acid. Use of a monovalent construct according to any one of the preceding claims.
Aspect F-147: Monovalent construct consists essentially of SEQ ID NO: 5, and comprises one or more (eg, 2, 3, 4, 5, 6, 7, 8, 9, or 10 , 11 or 12) amino acids residues are mutated from Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly selected from Gly54Ap. Use of the monovalent construct according to any one of the aspects F-99 to F-101 in preparing a trivalent polypeptide, wherein the glutamic acid at position is changed to aspartic acid.
Aspect F-148: Monovalent construct consisting essentially of SEQ ID NO: 5, wherein the amino acid residues are Val5Leu, Ala14Pro, Ser19R, Ile20Leu, Glu44Gly, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, GlnGle54, GlnGLe. Use of the monovalent construct according to embodiment F-147, in preparing a trivalent polypeptide, wherein the glutamic acid at position 1 has been changed to aspartic acid.
Aspect F-149: monovalent construct consists essentially of SEQ ID NO: 5, wherein one or more (eg, 2, 3, 4, 5, 6, 7, or 8) amino acid residues are , Ser19R, Ile20Leu, Ala74Ser, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and a mutation selected from Gly54D, wherein the glutamic acid at position 1 has been changed to aspartic acid. Use of a monovalent construct according to aspect F-147.
Aspect F-150: monovalent construct consisting essentially of SEQ ID NO: 5, wherein one or more (eg, 2, 3, 4, or 5) amino acid residues is Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, And a mutation selected from Gly54D and wherein the glutamic acid at position 1 has been changed to aspartic acid. Use of a monovalent construct according to embodiment F-147, in preparing a trivalent polypeptide.
Aspect F-151: Monovalent construct consisting essentially of SEQ ID NO: 5, wherein the amino acid residue has been mutated in Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54D, changing glutamic acid at position 1 to aspartic acid Use of a monovalent construct according to embodiment F-150 in preparing a trivalent polypeptide.
Aspect F-152: One or more (eg, two, three, or four) amino acid residues are selected from Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu, wherein the monovalent construct consists essentially of SEQ ID NO: 5. Wherein the glutamic acid at position 1 has been changed to aspartic acid, wherein the monovalent construct according to any one of aspects F-99 to F-101, when preparing a trivalent polypeptide. use.
Aspect F-153: wherein the monovalent construct consists essentially of SEQ ID NO: 5, wherein one or more (eg, 2, 3, 4, 5, 6, or 7) amino acid residues are Ala14Pro, Aspect F-99 to preparing a trivalent polypeptide having undergone a mutation selected from Ser19Arg, Ile20Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu, wherein the glutamic acid at position 1 has been changed to aspartic acid. Use of a monovalent construct according to any one of F-101.
Aspect F-154: wherein the monovalent construct consists essentially of SEQ ID NO: 5, wherein the amino acid residue is
Glu1Asp,
Glu1Asp, Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Glu1Asp, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Glu1Asp, Gly78Leu, Ala83Arg, Asp85Glu, and Arg105Gln;
Glu1Asp, Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Glu1Asp, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Glu1Asp, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54Asp,
Glu1Asp and Gly54Asp,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Ala83Arg;
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Asp85Glu,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Arg105Gln,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Asp85Glu,
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Arg105Gln, Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Asp85Glu, and Arg105, Arg
Glu1Asp, Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, Asp85Glu, and Arg105Gln
Use of a monovalent construct according to embodiment F-147 in preparing a trivalent construct, which has been mutated.
Aspect F-155: For preparing a trivalent polypeptide having SEQ ID NO: 142, wherein the amino acid sequence having SEQ ID NO: 138 is linked to at least two additional amino acid sequences having SEQ ID NO: 5 via a 15GS linker. Use of the amino acid sequence having SEQ ID NO: 138.
Aspect F-156: In preparing a trivalent polypeptide having SEQ ID NO: 143, wherein the amino acid sequence having SEQ ID NO: 139 is linked to at least two additional amino acid sequences having SEQ ID NO: 62 via a 15GS linker. Use of the amino acid sequence having SEQ ID NO: 139.
Aspect F-157: In preparing a trivalent polypeptide having SEQ ID NO: 144, wherein the amino acid sequence having SEQ ID NO: 140 is linked via at least two further amino acid sequences having SEQ ID NO: 65 via a 15GS linker. Use of the amino acid sequence having SEQ ID NO: 140.
Aspect F-158: For preparing a trivalent polypeptide having SEQ ID NO: 145, wherein the amino acid sequence having SEQ ID NO: 141 is linked to at least two additional amino acid sequences having SEQ ID NO: 76 via a 15GS linker. Use of the amino acid sequence having SEQ ID NO: 141.
Aspect F-159: In preparing a trivalent polypeptide having SEQ ID NO: 162, wherein the amino acid sequence having SEQ ID NO: 154 is linked to at least two additional amino acid sequences having SEQ ID NO: 75 via a 15GS linker. Use of the amino acid sequence having SEQ ID NO: 154.
Aspect F-160: In preparing a trivalent polypeptide having SEQ ID NO: 163, wherein the amino acid sequence having SEQ ID NO: 155 is linked to at least two additional amino acid sequences having SEQ ID NO: 147 via a 15GS linker. Use of the amino acid sequence having SEQ ID NO: 155.
Aspect F-161: For preparing a trivalent polypeptide having SEQ ID NO: 164, wherein the amino acid sequence having SEQ ID NO: 156 is linked via at least two further amino acid sequences having SEQ ID NO: 149 via a 15GS linker. Use of the amino acid sequence having SEQ ID NO: 156.
Aspect F-162: In preparing a trivalent polypeptide having SEQ ID NO: 165, wherein the amino acid sequence having SEQ ID NO: 157 is linked to at least two additional amino acid sequences having SEQ ID NO: 153 via a 15GS linker. Use of the amino acid sequence having SEQ ID NO: 157.
Aspect E-262: Amino acid sequence itself or Aspect according to any one of Aspects A-1 to A-29, Aspects C-1 to C-38, and Aspects W-1 to W-38, respectively. B-1 to B-18, an increased half-life compared to the Nanobody (registered trademark) itself according to any one of aspects D-1 to D-38, and aspects X-1 to X-38, The polypeptide according to any one of aspects E-1 to E-261.
Aspect E-263: Any of Aspects A-1 to A-29, Aspects C-1 to C-38, and Aspects W-1 to W-38, wherein one or more other linking units respectively correspond. The amino acid sequence itself described in one, or Nanobody (registered trademark) according to any one of aspects B-1 to B-18, aspects D-1 to D-38, and aspects X-1 to X-38 The polypeptide according to aspect E-262, which provides a polypeptide having an increased half-life relative to itself.
Aspect E-264: One or more of the other binding units described above providing a polypeptide having an increased half-life is a serum protein or fragment thereof, a binding unit capable of binding a serum protein, an Fc portion, and a serum The polypeptide according to aspect E-262 or E-263, selected from the group consisting of small proteins or small peptides capable of binding to proteins.
Aspect E-265: Aspects E-262 to E-264, wherein said one or more other binding units providing a polypeptide having an increased half-life are selected from the group consisting of human serum albumin or a fragment thereof. The polypeptide according to any one of the above.
Aspect E-266: One or more of the other binding units described above that provide an increased half-life polypeptide can bind serum albumin (eg, human serum albumin) or serum immunoglobulin (eg, IgG). The polypeptide according to any one of aspects E-262 to E-265, selected from the group consisting of binding units.
Aspect E-267: One or more of the other binding units described above that provide a polypeptide with an increased half-life can bind serum albumin (eg, human serum albumin) or serum immunoglobulin (eg, IgG). A domain antibody, an amino acid sequence suitable for use as a domain antibody, a single domain antibody, an amino acid sequence suitable for use as a single domain antibody, a "dAb", an amino acid sequence suitable for use as a dAb, or a Nanobody ( (Registered trademark), the polypeptide according to any one of aspects E-262 to E-266.
Aspect E-268: One or more of the other binding units described above that provide a polypeptide with an increased half-life can bind serum albumin (eg, human serum albumin) or serum immunoglobulin (eg, IgG). The polypeptide according to any one of aspects E-262 to E-267, which is a Nanobody (R).
Aspect E-269: corresponding amino acid sequence according to any one of Aspects A-1 to A-29, Aspects C-1 to C-38, and Aspects W-1 to W-38, or an aspect B-1 to B-18, at least 1.5 times the half-life of Nanobody (registered trademark) itself according to any one of aspects D-1 to D-38 and aspects X-1 to X-38. The polypeptide according to any one of aspects E-262 to E-268, having a half-life, preferably at least 2-fold (eg at least 5-fold), such as at least 10-fold, or more than 20-fold greater.
Aspect E-270: Amino acid sequence itself or Aspect according to any one of Aspects A-1 to A-29, Aspects C-1 to C-38, and Aspects W-1 to W-38, respectively. Compared with Nanobody (registered trademark) itself according to any one of B-1 to B-18, Aspects D-1 to D-38, and Aspects X-1 to X-38, more than 1 hour, preferably Any one of embodiments E-262 to E-269, having an increased serum half-life of more than 2 hours, more preferably more than 6 hours (eg more than 12 hours), or even more than 24 hours, 48 hours or 72 hours. 2. The polypeptide according to item 1.
Aspect E-271: at least about 12 hours, preferably at least 24 hours, more preferably at least 48 hours, even more preferably at least 72 hours or more; for example, at least 5 days (eg, about 5-10 days), preferably at least 9 hours Days (eg, about 9-14 days), more preferably at least about 10 days (eg, about 10-15 days), or at least about 11 days (eg, about 11-16 days), more preferably at least about 12 days. Polymorph according to any one of aspects E-262 to E-270, having a serum half-life in humans of greater than (e.g., about 12-18 days) or more than 14 days (e.g., about 14-19 days). peptide.
Aspect G-1: the amino acid sequence of any one or more of aspects A-1 to A-29, aspects C-1 to C-38, and aspects W-1 to W-38, and / or Or Nanobody (registered trademark) according to any one or more of aspects B-1 to B-18, aspects D-1 to D-38, and aspects X-1 to X-38, and / or 1 comprising or consisting essentially of a polypeptide according to any one or more of embodiments E-1 to E-271, optionally linked via one or more linkers. A compound or construct optionally further comprising one or more other groups, residues, moieties or binding units.
Aspect G-2: Compound or construct according to aspect G-1, wherein the one or more other groups, residues, moieties or binding units are amino acid sequences.
Aspect G-3: Compound or construct according to aspect G-1 or G-2, which is one or more amino acid sequences when one or more linkers described above are present.
Aspect G-4: Compound or construct according to any one of aspects G-1 to G-3, wherein said one or more other groups, residues, moieties or binding units are immunoglobulin sequences.
Aspect G-5: wherein one or more of the other groups, residues, moieties or binding units described above comprises an amino acid sequence suitable for use as a domain antibody, a domain antibody, a single domain antibody, a single domain antibody. Aspect G-1 to G-4, selected from the group consisting of an amino acid sequence suitable for use, "dAb", an amino acid sequence suitable for use as dAb, or Nanobody (registered trademark). Or a compound of the formula:
Aspect G-6: Compound or construct according to any one of aspects G-1 to G-5, wherein one or more of the above amino acid sequences of the invention is an immunoglobulin sequence.
Aspect G-7: One or more of the above amino acid sequences of the invention is a domain antibody, an amino acid sequence suitable for use as a domain antibody, a single domain antibody, an amino acid sequence suitable for use as a single domain antibody , “DAb”, an amino acid sequence suitable for use as a dAb, or a compound or construct according to any one of aspects G-1 to G-6, selected from the group consisting of Nanobodies®.
Aspect G-8: One or more Nanobodies® according to any one of Aspects B-1 to B-18, Aspects D-1 to D-38, and Aspects X-1 to X-38 Any one of aspects G-1 to G-7, wherein the one or more other groups, residues, moieties, or linking units comprise or consist essentially of a Nanobody®. A compound or construct according to any one of the preceding claims.
Aspect G-9: Compound or construct according to any one of aspects G-1 to G-8, which is a multivalent construct.
Aspect G-10: Compound or construct according to any one of aspects G-1 to G-9, which is a multispecific construct.
Aspect G-11: Amino acid sequence itself or Aspect according to any one of Aspects A-1 to A-29, Aspects C-1 to C-38, and Aspects W-1 to W-38, respectively, or Aspect Nanobody (registered trademark) itself according to any one of B-1 to B-18, Aspects D-1 to D-38, and Aspects X-1 to X-38, or Aspects E-1 to E-271 The compound or construct according to any one of the aspects G-1 to G-10, wherein the half life is increased compared to the polypeptide according to any one of the above.
Aspect G-12: Aspects A-1 to A-29, Aspects C-1 to C-38, and Aspect W-1 wherein one or more other groups, residues, moieties or linking units respectively correspond. To the amino acid sequence itself according to any one of Embodiments W-1 to W-38, or any one of Embodiments B-1 to B-18, Embodiments D-1 to D-38, and Embodiments X-1 to X-38 Embodiment G-1 which provides a compound or construct having an increased half-life compared to the described Nanobody® itself or the polypeptide itself according to any one of embodiments E-1 to E-271. The compound or construct according to any one of G-11.
Aspect G-13: One or more of the other groups, residues, moieties or binding units described above that provide compounds or constructs with increased half-life can bind serum proteins or fragments thereof, serum proteins The compound or construct of aspect G-12, wherein the compound or construct is selected from the group consisting of a binding unit, an Fc moiety, and a small protein or small peptide capable of binding a serum protein.
Aspect G-14: One or more of the other groups, residues, moieties or binding units described above that provides a compound or construct with increased half-life is selected from the group consisting of human serum albumin or a fragment thereof. The compound or construct according to aspects G-12 to G-13.
Aspect G-15: One or more of the other groups, residues, moieties or binding units described above that provide a compound or construct with an increased half-life is a serum albumin (eg, human serum albumin) or a serum immunoglobulin (eg, The compound or construct according to any one of aspects G-12 to G-14, wherein the compound or construct is selected from the group consisting of a binding unit capable of binding to IgG).
Aspect G-16: One or more of the other groups, residues, moieties or binding units described above that provide a compound or construct with an increased half-life is a serum albumin (eg, human serum albumin) or a serum immunoglobulin (eg, IgG) capable of binding to a domain antibody, an amino acid sequence suitable for use as a domain antibody, a single domain antibody, an amino acid sequence suitable for use as a single domain antibody, "dAb", for use as a dAb The compound or construct according to any one of aspects G-12 to G-15, selected from the group consisting of a suitable amino acid sequence or Nanobody®.
Aspect G-17: One or more of the other groups, residues, moieties or binding units described above that provide a compound or construct with an increased half-life is a serum albumin (eg, human serum albumin) or a serum immunoglobulin (eg, The compound or construct according to any one of aspects G-12 to G-16, which is a Nanobody® capable of binding to IgG).
Aspect G-18: The corresponding amino acid sequence according to any one of Aspects A-1 to A-29, Aspects C-1 to C-38, and Aspects W-1 to W-38, or an aspect, respectively. Nanobody (registered trademark) itself according to any one of B-1 to B-18, Aspects D-1 to D-38, and Aspects X-1 to X-38, or Aspects E-1 to E-271 Having a half-life that is at least 1.5 times, preferably at least 2 times (eg, at least 5 times), eg, at least 10 times, or more than 20 times greater than the half life of the polypeptide according to any one of the above. The compound or construct according to any one of G-11 to G-17.
Aspect G-19: The corresponding amino acid sequence according to any one of Aspects A-1 to A-29, Aspects C-1 to C-38, and Aspects W-1 to W-38, or an aspect, respectively. Nanobody (registered trademark) itself according to any one of B-1 to B-18, Aspects D-1 to D-38, and Aspects X-1 to X-38, or Aspects E-1 to E-271 Or more than 1 hour, preferably more than 2 hours, more preferably more than 6 hours (eg more than 12 hours) or even more than 24 hours, 48 hours or 72 hours compared to the polypeptide itself according to any one of The compound or construct according to any one of aspects G-11 to G-18, having an increased serum half-life.
Aspect G-20: at least about 12 hours, preferably at least 24 hours, more preferably at least 48 hours, even more preferably at least 72 hours or more; for example at least 5 days (eg, about 5-10 days), preferably at least 9 hours Days (eg, about 9-14 days), more preferably at least about 10 days (eg, about 10-15 days), or at least about 11 days (eg, about 11-16 days), more preferably at least about 12 days. A compound according to any one of aspects G-11 to G-19, having a serum half-life in humans of greater than (e.g. about 12-18 days) or more than 14 days (e.g. about 14-19 days). Or a construct.

Aspect M-1: Amino acid sequence according to any one of Aspects A-1 to A-29, Aspects C-1 to C-30, and Aspects W-1 to W-38, Aspects B-1 to B- 18. Nanobody (registered trademark) according to any one of aspects D-1 to D-30 and aspects X-1 to X-38, and Nanobody (registered trademark) according to any one of aspects E-1 to E-271. Polypeptide, compound or construct according to any one of aspects G-1 to G-20 (as obtained by expression of a nucleic acid or nucleotide sequence encoding it), or aspect F-1 A nucleic acid or nucleotide sequence encoding the monovalent construct according to any one of to F-4.
Aspect M-2: Nucleic acid or nucleotide sequence according to Aspect M-1, which is in the form of a genetic construct.
Aspect M-3: Aspects according to any one of aspects F-1 to F-4 for preparing a genetic construct encoding a multivalent polypeptide according to any one of aspects E-1 to E-271. Use of a nucleic acid or nucleotide sequence according to aspect M-1, which encodes a monovalent construct
Aspect M-4: Use of a nucleic acid or nucleotide sequence according to aspect M-2, wherein the genetic construct encodes a multivalent (eg, bivalent) construct.
Aspect N-1: Amino acid sequence according to any one of Aspects A-1 to A-29, Aspects C-1 to C-38, and Aspects W-1 to W-38, Aspects B-1 to B- 18. Nanobody (registered trademark) according to any one of aspects D-1 to D-38 and aspects X-1 to X-38, and Nanobody (registered trademark) according to any one of aspects E-1 to E-271. Polypeptide, compound or construct according to any one of aspects G-1 to G-20 (as obtained by expression of a nucleic acid or nucleotide sequence encoding it), or aspect F-1 F-4, or can be expressed under suitable circumstances; and / or the nucleic acid or nucleotide sequence according to embodiment M-1 or M-2 is A host or host cell.
Aspect O-1: Amino acid sequence according to any one of Aspects A-1 to A-29, Aspects C-1 to C-38, and Aspects W-1 to W-38, Aspects B-1 to B- 18. Nanobody (registered trademark) according to any one of aspects D-1 to D-38 and aspects X-1 to X-38, and Nanobody (registered trademark) according to any one of aspects E-1 to E-271. Polypeptide, compound or construct according to any one of aspects G-1 to G-20, monovalent construct according to any one of aspects F-1 to F-4, or aspect M-1 or M A composition comprising at least one nucleic acid or nucleotide sequence according to -2.
Aspect O-2: The composition according to aspect O-1, which is a pharmaceutical composition.
Aspect O-3: Pharmaceutical composition, further comprising at least one pharmaceutically acceptable carrier, diluent or excipient, and / or adjuvant, and optionally one or more further pharmaceutically The composition according to aspect O-1 or O-2, comprising an active polypeptide and / or compound.
Aspect P-1: Amino acid sequence according to any one of Aspects A-1 to A-29, Aspects C-1 to C-38, and Aspects W-1 to W-38, Aspects B-1 to B- 18. Nanobody (registered trademark) according to any one of aspects D-1 to D-38 and aspects X-1 to X-38, and Nanobody (registered trademark) according to any one of aspects E-1 to E-271. Polypeptide, compound or construct according to any one of aspects G-1 to G-20 (as obtained by expression of a nucleic acid or nucleotide sequence encoding it), or aspect F-1 A method for producing the monovalent construct according to any one of -F-4, or the composition according to any one of aspects O-1 to O-3,
a) expressing the nucleic acid or nucleotide sequence according to embodiment M-1 or M-2 in a suitable host cell or host organism or in another suitable expression system, optionally followed by
b) The amino acid sequence according to any one of Aspects A-1 to A-29, Aspects C-1 to C-38, and Aspects W-1 to W-38 thus obtained, Aspect B -1 to B-18, Nanobody (registered trademark) according to any one of Embodiments D-1 to D-38, and Embodiments X-1 to X-38, and any one of Embodiments E-1 to E-271. A polypeptide according to one, the compound or construct according to any one of aspects G-1 to G-20 (as obtained by expression of a nucleic acid or nucleotide sequence encoding it), Or a step of isolating and / or purifying the monovalent construct according to any one of aspects F-1 to F-4
At least comprising:
Aspect P-2: Amino acid sequence according to any one of Aspects A-1 to A-29, Aspects C-1 to C-38, and Aspects W-1 to W-38, Aspects B-1 to B- 18. Nanobody (registered trademark) according to any one of aspects D-1 to D-38 and aspects X-1 to X-38, and Nanobody (registered trademark) according to any one of aspects E-1 to K-271. Polypeptide, compound or construct according to any one of aspects G-1 to G-20 (as obtained by expression of a nucleic acid or nucleotide sequence encoding it), or aspect F-1 A method for producing the monovalent construct according to any one of -F-4, or the composition according to any one of aspects O-1 to O-3,
a) The host or host cell according to Aspect N-1 is at least one of Aspects A-1 to A-29, Aspects C-1 to C-38, and Aspects W-1 to W-38. Nanobody (registered trademark) according to any one of aspects B-1 to B-18, aspects D-1 to D-38, and aspects X-1 to X-38, aspect E -1 to E-271, the compound or construct according to any one of aspects G-1 to G-20 (obtained by expression of a nucleic acid or nucleotide sequence encoding the same). Or the monovalent construct according to any one of aspects F-1 to F-4, or the composition according to any one of aspects O-1 to O-3. The above-mentioned host or the above-mentioned host cell is cultured and cultured under conditions such as expression and / or production. / Or the step of maintaining, then optionally
b) The amino acid sequence according to any one of Aspects A-1 to A-29, Aspects C-1 to C-38, and Aspects W-1 to W-38 thus obtained, Aspect B -1 to B-18, Nanobody (registered trademark) according to any one of Embodiments D-1 to D-38, and Embodiments X-1 to X-38, and any one of Embodiments E-1 to E-271. A polypeptide according to one, the compound or construct according to any one of aspects G-1 to G-20 (as obtained by expression of a nucleic acid or nucleotide sequence encoding it), Or a step of isolating and / or purifying the monovalent construct according to any one of aspects F-1 to F-4, or the composition according to any one of aspects O-1 to O-3.
At least comprising:
Aspect P-3: A method for preparing a divalent or trivalent polypeptide according to any one of aspects E-7 to E-261, wherein two or more monovalent amino acid sequences or aspect F-1 A method comprising at least linking the monovalent construct according to any one of to F-4 to, for example, one or more linkers.
Aspect P-4:
a) two or more nucleic acid sequences according to aspect M-1 encoding a monovalent construct according to any one of aspects F-1 to F-4 (also for example a nucleic acid encoding one or more linkers) And further linking one or more additional elements of the gene construct known per se) to obtain the gene construct according to aspect M-2,
b) expressing the gene construct obtained in a) in a suitable host cell or organism or in another suitable expression system, optionally followed by
c) a step of isolating and / or purifying the divalent or trivalent polypeptide according to any one of aspects E-7 to E-261 thus obtained.
The method of embodiment P-3, comprising:
Aspect Q-1: A method for screening for an amino acid sequence having directivity to protein F of hRSV,
a. Preparing a set, collection or library of nucleic acid sequences encoding amino acid sequences;
b. It is capable of binding to and / or has an affinity for the envelope protein of the virus, and is a Nanobody® of the invention, such as SEQ ID NO: 60 to SEQ ID NO: 76, SEQ ID NO: 138 to SEQ ID NO: 141, and SEQ ID NO: 146 to SEQ ID NO: 157 (Table A-4), or a polypeptide or construct comprising at least one Nanobody (R) of the invention, e.g., SEQ ID NO: 77 to SEQ ID NO: 99, SEQ ID NO: 142 to sequence No. 145 and a nucleic acid sequence encoding an amino acid sequence that is cross-blocked by or cross-blocks with a polypeptide or construct comprising at least one of SEQ ID NOs: 158-165 (see Table A-5) Screening the set, collection or library of nucleic acid sequences for In beauty
c. Isolating the nucleic acid sequence and then expressing the amino acid sequence, isolating
At least comprising:
Aspect R-1: A method for preventing and / or treating hRSV infection, comprising a pharmaceutically active amount of at least one of aspects A-1 to A-29, aspects C-1 to C-30, and Amino acid sequence according to any one of aspects W-1 to W-38, any one of aspects B-1 to B-18, aspects D-1 to D-38, and aspects X-1 to X-38 Nanobody (registered trademark), polypeptide according to any one of aspects E-1 to E-271, compound or construct according to any one of aspects G-1 to G-20, aspect A monovalent construct according to any one of F-1 to F-4 and / or a composition according to any one of aspects O-1 to O-3, to a subject in need thereof. Administering, comprising: administering.
Aspect R-2: at least one of respiratory disease, upper respiratory tract infection, lower respiratory tract infection, bronchiolitis (inflammation of the peripheral airways in the lungs), pneumonia, dyspnea, cough, (recurrent) wheezing and asthma A method of preventing and / or treating one of the above, wherein a pharmaceutically active amount of at least one of embodiments A-1 to A-29, embodiments C-1 to C-38, and embodiments W-1 to W-. 38. The Nanobody according to any one of Aspects B-1 to B-18, Aspects D-1 to D-38, and Aspects X-1 to X-38 (registered Trademark), polypeptide according to any one of aspects E-1 to E-271, compound or construct according to any one of aspects G-1 to G-20, aspects F-1 to F-4 And / or the monovalent construct according to any one of aspects O-1 to O-3. The composition, comprising administering to a subject in need thereof.
Aspect R-3: Amino acid sequence according to any one of Aspects A-1 to A-29, Aspects C-1 to C-38, and Aspects W-1 to W-38, Aspects B-1 to B- 18. Nanobody (registered trademark) according to any one of aspects D-1 to D-38 and aspects X-1 to X-38, and Nanobody (registered trademark) according to any one of aspects E-1 to E-271. Polypeptide, compound or construct according to any one of aspects G-1 to G-20, monovalent construct according to any one of aspects F-1 to F-4, and / or aspect O-1 Preventing and / or treating at least one disease or disorder that can be prevented and / or treated by administering a composition according to any one of -O-3 to a subject in need thereof. A method, comprising a pharmaceutically active amount of at least one embodiment A-1 to A-29, embodiment -1 to C-38, and the amino acid sequence according to any one of aspects W-1 to W-38, aspects B-1 to B-18, aspects D-1 to D-38, and aspect X-1 -N-body (registered trademark) according to any one of aspects X-1 to X-38, polypeptide according to any one of aspects E-1 to E-271, or any one of aspects G-1 to G-20 A compound or construct according to any one of aspects F-1 to F-4, and / or a composition according to any one of aspects O-1 to O-3, A method comprising administering to a subject in need thereof.
Aspect R-4: A method for immunotherapy, comprising a pharmaceutically active amount of at least one of aspects A-1 to A-29, aspects C-1 to C-38, and aspects W-1 to W-. 38. The Nanobody according to any one of Aspects B-1 to B-18, Aspects D-1 to D-38, and Aspects X-1 to X-38 (registered Trademark), polypeptide according to any one of aspects E-1 to E-271, compound or construct according to any one of aspects G-1 to G-20, aspects F-1 to F-4 Administering a monovalent construct according to any one of the preceding, and / or a composition according to any one of the embodiments O-1 to O-3 to a subject in need thereof. .
Aspect R-5: for use in the preparation of a pharmaceutical composition for the prevention and / or treatment of hRSV infection and / or in one or more of the methods according to aspects R-1 to R-4. Amino acid sequence according to any one of aspects A-1 to A-29, aspects C-1 to C-38, and aspects W-1 to W-38, aspects B-1 to B-18, aspect D -1 to D-38, and Nanobody (registered trademark) according to any one of aspects X-1 to X-38, polypeptide according to any one of aspects E-1 to E-271, aspect Compound or construct according to any one of G-1 to G-20, monovalent construct according to any one of aspects F-1 to F-4, and / or aspects O-1 to O-3 Use of a composition according to any one of the preceding claims.
Aspect R-6: at least one of respiratory disease, upper respiratory tract infection, lower respiratory tract infection, bronchiolitis (inflammation of the peripheral airways in the lungs), pneumonia, dyspnea, cough, (recurrent) wheezing and asthma The amino acid sequence according to any one of aspects A-1 to A-29, aspects C-1 to C-38, and aspects W-1 to W-38 for prevention and / or treatment, aspect B- Nanobody (registered trademark) according to any one of Embodiments 1 to B-18, Embodiments D-1 to D-38, and Embodiments X-1 to X-38, and any one of Embodiments E-1 to E-271. The compound according to any one of aspects G-1 to G-20, the monovalent construct according to any one of aspects F-1 to F-4, and / or The composition according to any one of aspects O-1 to O-3.
Aspect S-1: Amino acid sequence according to any one of Aspects A-1 to A-29, Aspects C-1 to C-38, and Aspects W-1 to W-38, Aspects B-1 to B- 18. Nanobody (registered trademark) according to any one of aspects D-1 to D-38 and aspects X-1 to X-38, and / or any one of aspects E-1 to E-271 2. A portion or fragment of the polypeptide according to 1.
Aspect S-2: The moiety according to aspect S-1, which specifically binds to antigenic site II on protein F of hRSV and / or competes with SYNAGIS® for binding of hRSV to protein F or fragment.
Aspect S-3: Dissociation constant (K of 1000 nM to 1 nM or less, preferably 100 nM to 1 nM or less, more preferably 10 nM to 1 nM or less _D ) The part or fragment according to aspect S-1 or S-2, which is capable of specifically binding to protein F of hRSV in (1).
Aspect S-4: 10 ⁴ M ^-1 s ^-1 ~ About 10 ⁷ M ^-1 s ^-1 , Preferably 10 ⁵ M ^-1 s ^-1 -10 ⁷ M ^-1 s ^-1 , More preferably about 10 ⁶ M ^-1 s ^-1 More than k _on The portion or fragment of any one of aspects S-2 or S-3, which is capable of binding specifically to protein F of hRSV at a rate.
Aspect S-5: 10 ^-2 s ^-1 (T _1/2 = 0.69 s) to 10 ^-4 s ^-1 (T _1/2 Is a few days), preferably 10 ^-3 s ^-1 -10 ^-4 s ^-1 The following k _off The portion or fragment of any one of aspects S-2 to S-4, which is capable of binding specifically to protein F of hRSV at a rate.
Aspect S-6: comprising or consisting essentially of a portion or fragment according to any one or more of aspects S-1 to S-5, optionally via one or more linkers. Or a compound or construct optionally further comprising one or more other groups, residues, moieties or linking units linked together.
Aspect S-7: Compound or construct according to aspect S-6, wherein the one or more other groups, residues, moieties or binding units is an amino acid sequence.
Aspect S-8: Compound or construct according to aspect S-6 or S-7, wherein one or more of the above linkers, when present, is one or more amino acid sequences.
Aspect S-9: Nucleic acid encoding a portion or fragment according to any one of aspects S-1 to S-5, or a compound or construct according to any one of aspects S-6 to S-8 Nucleotide sequence.
Aspect S-10: Part or fragment according to any one of aspects S-1 to S-5, compound or construct according to any one of aspects S-6 to S-8, or aspect S-9 A composition comprising at least one nucleic acid or nucleotide sequence described in 1.
Aspect T-1: Amino acid sequence according to any one of Aspects A-1 to A-29, Aspects C-1 to C-38, and Aspects W-1 to W-38, or Aspects B-1 to B-B -18, the derivative of Nanobody (registered trademark) according to any one of aspects D-1 to D-38 and aspects X-1 to X-38.
Aspect T-2: Aspect T-1 that is capable of specifically binding to antigenic site II on protein F of hRSV and / or competes with SYNAGIS® for binding of hRSV to protein F Derivatives.
Aspect T-3: 10 ⁴ M ^-1 s ^-1 ~ About 10 ⁷ M ^-1 s ^-1 , Preferably 10 ⁵ M ^-1 s ^-1 -10 ⁷ M ^-1 s ^-1 , More preferably about 10 ⁶ M ^-1 s ^-1 More than k _on The derivative according to aspect T-1 or T-2, which is capable of binding specifically to protein F of hRSV at a rate.
Aspect T-4: 10 ⁴ M ^-1 s ^-1 ~ About 10 ⁷ M ^-1 s ^-1 , Preferably 10 ⁵ M ^-1 s ^-1 -10 ⁷ M ^-1 s ^-1 , More preferably about 10 ⁶ M ^-1 s ^-1 More than k _on The derivative according to aspect T-2 or T-3, which is capable of binding specifically to protein F of hRSV at a rate.
Aspect T-5: 10 ^-2 s ^-1 (T _1/2 = 0.69 s) to 10 ^-4 s ^-1 (T _1/2 Is a few days), preferably 10 ^-3 s ^-1 -10 ^-4 s ^-1 The following k _off The derivative according to any one of aspects T-2 to T-4, which is capable of binding specifically to protein F of hRSV at a rate.
Aspect T-6: Compound or construct according to any one of aspects G-1 to G-20, or a derivative of the polypeptide according to any one of aspects E-1 to E-271.
Aspect T-7: Aspect T-6, which is capable of binding specifically to antigenic site II on protein F of hRSV and / or competes with SYNAGIS® for binding of hRSV to protein F. Derivatives.
Aspect T-8: Dissociation constant (K) of 100 nM to 0.1 nM or less, preferably 10 nM to 0.1 nM or less, more preferably 1 nM to 0.1 nM or less _D The derivative according to aspect T-6 or T-7, which is capable of specifically binding to protein F of hRSV in (1).
Aspect T-9: 10 ⁴ M ^-1 s ^-1 ~ About 10 ⁷ M ^-1 s ^-1 , Preferably 10 ⁵ M ^-1 s ^-1 -10 ⁷ M ^-1 s ^-1 , More preferably about 10 ⁶ M ^-1 s ^-1 More than k _on The derivative according to aspects T-6 to T-8, which is capable of binding specifically to protein F of hRSV at a rate.
Aspect T-10: 10 ^-2 s ^-1 (T _1/2 = 0.69 s) to 10 ^-4 s ^-1 (T _1/2 Is a few days), preferably 10 ^-3 s ^-1 -10 ^-4 s ^-1 , More preferably 5 × 10 ^-3 s ^-1 -10 ^-4 s ^-1 The following k _off The derivative according to any one of aspects T-6 to T-9, which is capable of binding specifically to protein F of hRSV at a rate.
Embodiment T-11: With an IC50 value of 10 pM to 1000 pM, preferably 10 pM to 250 pM, more preferably 50 pM to 200 pM or less, for example as determined in a microneutralization assay for the hRSV strain Long (for example as described in Example 6). The derivative according to any one of aspects T-6 to T-10, which is capable of neutralizing hRSV as described above.
Aspect T-12: at least the same, preferably better, at least 10 times better, preferably 20 times better, more preferably 50 times better as compared to the IC50 value obtained with SYNAGIS® Neutralizes hRSV in a microneutralization assay, e.g., with respect to RSV strain Long (e.g., as described in Example 6) with an IC50 value that is even more preferably 60-fold, 70-fold, 80-fold or more than 80-fold better. The derivative according to any one of aspects T-6 to T-11, which is capable of:
Aspect T-13: corresponding amino acid sequence according to any one of Aspects A-1 to A-29, Aspects C-1 to C-38, and Aspects W-1 to W-38, Aspect B -1 to B-18, Nanobody (registered trademark) itself according to any one of Embodiments D-1 to D-38, and Embodiments X-1 to X-38, and any of Embodiments E-1 to E-271. The polypeptide according to any one of the aspects, the compound or construct itself according to any one of aspects G-1 to G-20, or the monovalent construct according to any one of aspects F-1 to F-4 Any of embodiments T-1 to T-12, having a half-life at least 1.5 times, preferably at least 2 times (eg at least 5 times), for example at least 10 times, or more than 20 times greater than its own half life. A derivative according to one of the above.
Aspect T-14: The corresponding amino acid sequence according to any one of Aspects A-1 to A-29, Aspects C-1 to C-38, and Aspects W-1 to W-38, Aspect B, respectively -1 to B-18, Nanobody (registered trademark) itself according to any one of Embodiments D-1 to D-38, and Embodiments X-1 to X-38, and any of Embodiments E-1 to E-271. The polypeptide according to any one of the aspects, the compound or construct itself according to any one of aspects G-1 to G-20, or the monovalent construct according to any one of aspects F-1 to F-4 Aspect T having an increased serum half-life of more than 1 hour, preferably more than 2 hours, more preferably more than 6 hours (eg more than 12 hours), or even more than 24 hours, 48 hours or 72 hours compared to itself The derivative according to any one of -1 to T-13.
Aspect T-15: at least about 12 hours, preferably at least 24 hours, more preferably at least 48 hours, even more preferably at least 72 hours or more; for example at least 5 days (eg, about 5 to 10 days), preferably at least 9 hours Days (eg, about 9-14 days), more preferably at least about 10 days (eg, about 10-15 days), or at least about 11 days (eg, about 11-16 days), more preferably at least about 12 days. The derivative according to any one of aspects T-1 to T-14, having a serum half-life in humans of greater than (e.g., about 12 to 18 days or more), or more than 14 days (e.g., about 14 to 19 days). .
Aspect T-16: The derivative according to any one of aspects T-1 to T-15, which is a pegylated derivative.
Aspect T-17: Including or consisting essentially of a derivative according to any one or more of aspects T-1 to T-16, optionally linked via one or more linkers Or a compound or construct optionally further comprising one or more other groups, residues, moieties or linking units.
Aspect T-18: Compound or construct according to aspect T-17, wherein said one or more other groups, residues, moieties or binding units is an amino acid sequence.
Aspect T-19: Compound or construct according to aspect T-17 or T-18, which is one or more amino acid sequences when one or more linkers as described above are present.
Aspect T-20: Nucleic acid or nucleotide sequence encoding a derivative according to any one of aspects T-1 to T-16, or a compound or construct according to any one of aspects T-17 to T-19 .
Aspect T-21: Derivative according to any one of aspects T-1 to T-16, compound or construct according to any one of aspects T-17 to T-19, or as described in aspect T-20 A composition comprising at least one nucleic acid or nucleotide sequence of
Aspect U-1: An effective amount of the amino acid sequence according to any one of claims A-1 to A-29, claims C-1 to C-38, and claims W-1 to W-38. Nanobody (registered trademark) according to any one of claims B-1 to B-18, claims D-1 to D-38, and claims X-1 to X-38, claims E-1 to E -271, a compound or construct according to any one of claims G-1 to G-20, and / or a compound according to any one of claims F-1 to F-4. A method for administering a monovalent construct according to any one of claims 1 to 4 and / or a composition containing the same, comprising: A-1 to A-29, C-1 to C-38, and W-1. To B-38, Claims B-1 to B-18, Claims D-1 to D-38, and Claims X-1 to X-. No. 8, Nanobody (registered trademark) according to any one of claims E-1 to E-271, polypeptide according to any one of claims G-1 to G-20 A method comprising administering to the lung tissue a compound or construct according to claim 1 and / or a monovalent construct according to any one of claims F-1 to F-4, and / or a composition comprising it.
Aspect U-2: Amino acid sequence according to any one of claims A-1 to A-29, claims C-1 to C-38, and claims W-1 to W-38, claim B- Nanobody (registered trademark) according to any one of Claims 1 to B-18, Claims D-1 to D-38, and Claims X-1 to X-38, Claims E-1 to E-271. The polypeptide according to any one of the claims, the compound or construct according to any one of claims G-1 to G-20, and / or the compound according to any one of claims F-1 to F-4. The method according to aspect U-1, wherein the monovalent construct, and / or a composition comprising the same, is administered by use of an inhaler, an intranasal delivery device or an aerosol.
Aspect U-3: Claim A of at least 5%, preferably at least 10%, 20%, 30%, 40%, more preferably at least 50%, 60%, 70%, even more preferably at least 80% or more. -1 to A-29, Claims C-1 to C-38, and any one of Claims W-1 to W-38, Claims B-1 to B-18, Claims Nanobody (registered trademark) according to any one of claims D-1 to D-38 and claims X-1 to X-38, and a polybody according to any one of claims E-1 to E-271. Peptides, compounds or constructs according to any one of claims G-1 to G-20, and / or monovalent constructs according to any one of claims F-1 to F-4, and / or The composition comprising it for at least 24 hours, preferably at least 48 hours, Preferably stable in at least 72 hours lung tissue, the method according to aspect U-1 or U-2 Ri.
Aspect U-4: Amino acid sequence according to any one of claims A-1 to A-29, claims C-1 to C-38, and claims W-1 to W-38, claim B- Nanobody (registered trademark) according to any one of Claims 1 to B-18, Claims D-1 to D-38, and Claims X-1 to X-38, Claims E-1 to E-271. The polypeptide according to any one of the claims, the compound or construct according to any one of claims G-1 to G-20, and / or the compound according to any one of claims F-1 to F-4. The method according to any one of aspects U-1 to U-3, wherein the monovalent construct, and / or the composition comprising it, is applied in pure form, ie when it is a liquid or a dry powder.
Aspect U-5: The amino acid sequence according to any one of claims A-1 to A-29, claims C-1 to C-38, and claims W-1 to W-38, claim B- Nanobody (registered trademark) according to any one of Claims 1 to B-18, Claims D-1 to D-38, and Claims X-1 to X-38, Claims E-1 to E-271. The polypeptide according to any one of the claims, the compound or construct according to any one of claims G-1 to G-20, and / or the compound according to any one of claims F-1 to F-4. A monovalent construct, and / or a composition containing the same, according to any one of claims A-1 to A-29, claims C-1 to C-38, and claims W-1 to W-38 The amino acid sequence according to any one of claims B-1 to B-18, claims D-1 to D-38, and claims X-1 to X-38. A body (registered trademark), a polypeptide according to any one of claims E-1 to E-271, a compound or construct according to any one of claims G-1 to G-20, and / or Aspects U-1 to U-, wherein the composition is administered to lung tissue as a composition or formulation comprising the monovalent construct according to any one of claims F-1 to F-4 and a carrier suitable for pulmonary delivery. 3. The method according to any one of 3.
Aspect U-6: The amino acid sequence according to any one of claims A-1 to A-29, claims C-1 to C-38, and claims W-1 to W-38, claim B- Nanobody (registered trademark) according to any one of Claims 1 to B-18, Claims D-1 to D-38, and Claims X-1 to X-38, Claims E-1 to E-271. The polypeptide according to any one of the claims, the compound or construct according to any one of claims G-1 to G-20, and / or the compound according to any one of claims F-1 to F-4. And a carrier suitable for pulmonary delivery.
Aspect U-7: The amino acid sequence according to any one of claims A-1 to A-29, claims C-1 to C-38, and claims W-1 to W-38, claim B- Nanobody (registered trademark) according to any one of Claims 1 to B-18, Claims D-1 to D-38, and Claims X-1 to X-38, Claims E-1 to E-271. The polypeptide according to any one of the claims, the compound or construct according to any one of claims G-1 to G-20, and / or the compound according to any one of claims F-1 to F-4. A pharmaceutical device suitable for pulmonary delivery of a monovalent construct of the present invention and / or for use of a composition comprising the same.
Aspect U-8: Amino acid sequence according to any one of claims A-1 to A-29, claims C-1 to C-38, and claims W-1 to W-38, claim B- Nanobody (registered trademark) according to any one of Claims 1 to B-18, Claims D-1 to D-38, and Claims X-1 to X-38, Claims E-1 to E-271. The polypeptide according to any one of the claims, the compound or construct according to any one of claims G-1 to G-20, and / or the compound according to any one of claims F-1 to F-4. Pharmaceutical device according to aspect U-7, which is an inhaler for a liquid (e.g., a suspension of solid microparticles or droplets) comprising a monovalent construct.
Aspect U-9: The amino acid sequence according to any one of claims A-1 to A-29, claims C-1 to C-38, and claims W-1 to W-38, claim B- Nanobody (registered trademark) according to any one of Claims 1 to B-18, Claims D-1 to D-38, and Claims X-1 to X-38, Claims E-1 to E-271. The polypeptide according to any one of the claims, the compound or construct according to any one of claims G-1 to G-20, and / or the compound according to any one of claims F-1 to F-4. The pharmaceutical device according to aspect U-7, which is an aerosol comprising the monovalent construct of
Aspect U-10: The amino acid sequence according to any one of claims A-1 to A-29, claims C-1 to C-38, and claims W-1 to W-38, claim B- Nanobody (registered trademark) according to any one of Claims 1 to B-18, Claims D-1 to D-38, and Claims X-1 to X-38, Claims E-1 to E-271. The polypeptide according to any one of the claims, the compound or construct according to any one of claims G-1 to G-20, and / or the compound according to any one of claims F-1 to F-4. Pharmaceutical device according to aspect U-7, which is a dry powder inhaler comprising the monovalent construct in dry powder form.
Aspect U-11: A method for preventing and / or treating hRSV infection, comprising pharmaceutically active amounts of claims A-1 to A-29, claims C-1 to C-38, and claims. The amino acid sequence according to any one of Items W-1 to W-38, Claims B-1 to B-18, Claims D-1 to D-38, and Claims X-1 to X-38. Nanobody (registered trademark) according to any one of claims, polypeptide according to any one of claims E-1 to E-271, and polypeptide according to any one of claims G-1 to G-20. Administering the compound or construct and / or the monovalent construct according to any one of claims F-1 to F-4 and / or a composition comprising it to the lung tissue of a subject in need thereof A method, comprising:
Aspect U-12: at least one of respiratory disease, upper respiratory tract infection, lower respiratory tract infection, bronchiolitis (inflammation of the peripheral airways in the lungs), pneumonia, dyspnea, cough, (recurrent) wheezing and asthma A method of preventing and / or treating one of the claims, wherein a pharmaceutically active amount of claims A-1 to A-29, claims C-1 to C-38, and claims W-1 to W-. 38. The amino acid sequence according to any one of claims 38 to 31, B-1 to B-18, D-1 to D-38, and X-1 to X-38. Nanobody (registered trademark), the polypeptide according to any one of claims E-1 to E-271, the compound or construct according to any one of claims G-1 to G-20, and / or The monovalent construct according to any one of claims F-1 to F-4 and / or a composition comprising the same. Including administering to the pulmonary tissue of a subject in need, the method.
Aspect V-1: A method for preventing and / or treating hRSV infection, comprising the multivalent polypeptide according to any one of Aspects E-7 to E-261, and / or a pharmaceutical composition comprising the same. Administering a substance to a subject in need thereof.
Aspect V-2: Use of a multivalent polypeptide according to any one of aspects E-7 to E-261 for binding and / or neutralization of hRSV, and / or use of a pharmaceutical composition comprising the same.
Aspect V-3: Multivalent polypeptide according to any one of aspects E-7 to E-261 for binding and / or neutralization of various strains of hRSV, and / or pharmaceutical composition comprising same Use of things.
Aspect V-4: Multivalent polypeptide according to any one of aspects E-7 to E-261 for binding and / or neutralization of one or more escape variants of the virus, and / or Use of a pharmaceutical composition comprising
Aspect V-5: The method or use according to any one of aspects V-1 to V-4, wherein the multivalent polypeptide is divalent.
Aspect V-6: The method or use according to any one of aspects V-1 to V-4, wherein the multivalent polypeptide is trivalent.
Aspect V-7: Aspect V-1 to V-, wherein the multivalent polypeptide is administered according to any one of the methods according to claims U-1 to U-5 and / or U-11 to U-12. 6. The method or use according to any one of 6.
Aspect V-8: A method for preventing and / or treating an infection caused by an hRSV virus, wherein the pharmaceutically active amount of the multivalent polypeptide according to any one of Aspects E-7 to E-261. And / or administering a pharmaceutical composition comprising the same to a subject in need thereof.
Aspect V-9: Aspect V-8 wherein the multivalent compound or construct is selected from Table A-5 (SEQ ID NOs: 77-99, SEQ ID NOs: 138-141 and 146-157). The method described in.
Aspect V-10. A method according to aspect V-8 or V-9 for treating an infection with one or more RSV escape variants.
Aspect V-11: The method of aspect V-10, wherein the escape variant is an escape variant specific for antigenic site II.
Aspect V-12: Polyvalent compound or construct according to any one of the aspects E-7 to E-261 for binding and / or neutralization of one or more various escape variants of RSV, And / or use of a pharmaceutical composition comprising the same.
Aspect V-13: Use according to claim V-12, wherein the escape variant is an escape variant specific for antigenic site II.
Aspect V-14. The method of aspect V-8 or V-9 treating an infection with one or more strains of hRSV.
Aspect V-15: The method of aspect V-14, wherein the RSV strain is Long.
Aspect V-16: The method of aspect V-14, wherein the RSV strain is A-2.
Aspect V-17: The method of aspect V-14, wherein the RSV strain is B-1.
Aspect V-18: The method according to aspect V-14, wherein the multivalent polypeptide binds to and / or neutralizes Long and A-2 of the RSV strain.
Aspect V-19: The method according to aspect V-14, wherein the multivalent polypeptide binds to and / or neutralizes Long and B-1 of the RSV strain.
Aspect V-20: The method according to aspect V-14, wherein the multivalent polypeptide binds to and / or neutralizes RSV strain B-1 and A-2.
Aspect V-21: The method of aspect V-14, wherein the multivalent polypeptide binds to and / or neutralizes Long, A-2 and B-1 of the RSV strain.
Aspect V-22: Polyvalent compound or construct according to any one of aspects E-7 to E-261 for binding and / or neutralization of various strains of hRSV, and / or a pharmaceutical comprising same Use of the composition.
Aspect V-23: Use according to aspect V-22, wherein the strains of RSV are Long and A-2.
Aspect V-24: Use according to aspect V-22, wherein the strains of RSV are Long and B-1.
Aspect V-25: Use according to aspect V-22, wherein the strains of RSV are A-1 and B-1.
Aspect V-26: Use according to aspect V-22, wherein the strains of RSV are Long, A-2 and B-1.

Example 1:. To immunization 2 animals llama (156 and 157), according to standard protocols, hRSV F _TM- (lacking membrane anchor (Anchorless) form of a fusion protein, 70kDa; Corrall T. etal 2007, BMC Biotechnol. 7:17) for 6 boosts. Blood was collected from these animals 7 days after the sixth boost and 10 days after the sixth boost.

Two llamas (212 and 213) were immunized by intramuscular injection of 1 mg of RNA-inactivated RSV strain long A (Hytest (Turku, Finland); # 8 RSV79) followed by 0.5 mg of RSV. Four boosts were given with a biweekly regimen. Two llamas (206 and 207) were immunized by intramuscular injection with 1 mg of RNA-inactivated RSV strain long A, boosted two weeks later with 0.25 mg of RSV, followed by 50 μg of recombinant hRSV F. _{Three boosts with TM-} NN (fusion protein lacking membrane anchor, 70 kDa: Corral et al. 2007; BMC Biotechnol. 7:17) in a biweekly regimen. For all immunizations, the antigen was prepared as an oil-PBS emulsion with Stimune as adjuvant. Blood was collected from these animals 4 and 10 days after the fourth immunization, and lymph node biopsies were similarly collected 4 days after the fourth immunization. For the nanoclone procedure, 100 mL of blood was collected from llama 206 and llama 207 11 days after the last boost.

Example 2: Library construction Peripheral blood mononuclear cells were prepared from blood samples using Ficoll-Hypaque according to the manufacturer's instructions. Next, total RNA was extracted from these cells and lymph node arch cells and used as starting material for RT-PCR to amplify the gene fragment encoding Nanobody®. These fragments were cloned into a phagemid vector derived from pUC119 containing the LacZ promoter, E. coli phage pIII protein coding sequence, ampicillin or carbenicillin resistance gene, multiple cloning site and gen3 leader sequence. In frame with the Nanobody® coding sequence, the vector encodes a C-terminal c-myc tag and a (His) 6 tag. Phage were prepared and stored at 4 ° C. for further use to generate phage libraries 156, 157, 206, 207, 212 and 213 according to standard methods.

Example 3: by F protein hRSV To identify Nanobodies recognizing the fusion protein (TM) selected RSV Nanobody (TM), a library 156,157,206,207,212 and 213 _F TM- NN (Long fusion protein lacking a membrane anchor, 70 kDa; Corral T. et al. 2007, BMC Biotechnol. 7:17). _FTM- protein (25 ng / well) was immobilized on Nunc Maxisorp ELISA plates. Controls contained 0 μg / ml _FTM- . In the first and second selection, trypsin and SYNAGIS®: bound from F _TM- using (palivizumab, MedImmune, Zhao and Sullender 2005, J. Virol.79 3962 humanized monoclonal antibody according to) The phage was eluted. Remicade (Infliximab, anti-TNF; Centocor) was used as a control for SYNAGIS®. A 100 molar excess of Synagis® was used to identify Nanobodies® that specifically bind at the Synagis® binding site on RSV. The output of the first selection eluted with SYNAGIS® was used for the second selection.

Selection was performed using an inactivated hRSV strain Long (Hytest # 8 RSV79). _FTM- NN protein (25 ng / well) or RSV (5 μg / well to 50 μg / well) was immobilized on Nunc Maxisorp ELISA plates next to controls with 0 μg / ml antigen. In the first round of selection, the bound phage was converted to trypsin, Synagis® (palivizumab, a humanized monoclonal antibody, described in Zhao and Sullender 2005, J. Virol. 79: 396), or 101F Fab (International Publication No. No. 06/050280, was eluted from the _{F TM-} NN with the humanized monoclonal antibody). The output of the first selection eluted with SYNAGIS® or 101F Fab was converted to Numax Fab (motavizumab or MEDI-524, a third generation humanized monoclonal antibody product developed from palivizumab; WO 06) 050166), Synagis® or 101F Fab was used for elution and used for the second round of selection. Remicade (infliximab, anti-TNF, see WO 09/068625) was used as a control for Synagis®, and Omnitarg Fab (anti-Her2; in-house produced) was converted to Numax Fab and As a control for the 101F Fab. To identify Nanobodies® that specifically bind to antigenic site II or IV-VI epitopes on RSV F protein, a 100 molar excess of Synagis®, Numax Fab or 101F Fab was used. To obtain Nanobodies® specific for antigenic sites IV-VI, a second selection was performed with two biotinylated peptides: the first was a 101F binding epitope (Wu et al. 2007, J. Gen. Virol. 88: 2719-2723), a peptide containing residues 422 to 436 of the F protein (Long) (Abgent (San Diego, Calif.)) And a second peptidomimetic of the epitope of Mab19 (HWSISKPQ-PEG4). -K-biotin) (Chargelegue et al. 1998, J. Virol. 72: 2040-2056).

  The results from both selections were analyzed for enrichment factor (phages present in the eluate compared to controls). Based on these parameters, the best choice was chosen for further analysis. Individual colonies were picked and cultured in 96 deep well plates (1 mL volume) and Nanobody® expression was induced by adding IPTG. Periplasmic extracts (volume: about 80 μl) were prepared according to standard methods.

  To test selected clones in an RSV neutralization assay, periplasmic extracts from 10 ml cultures were partially purified using IMAC PhyTips (Phynexus Inc, San Jose, CA). Here, 800 μl of the periplasmic extract was loaded onto a Physics 200+ column pre-packed with immobilized metal affinity chromatography resin, followed by His-tagged Nanobody® with 30 μl of 0.1 M glycine-HCl / 0. Elution was carried out in 0.15 M NaCl (pH 3), after which the eluate was neutralized with 5 μl 0.5 M Tris-HCl (pH 8.5).

Example 4: Nanobody (R) Selection of RSV by _FTM- NN Using _Nanoclone Technology Peripheral blood mononuclear cells (PBMC) from a blood sample using Ficoll-Hypaque according to the manufacturer's instructions Was prepared. Antigen-specific B cells expressing heavy chain antibodies on their surface were isolated from PBMC by FACS sorting (for a description of nanoclone technology, see WO 06/079372). In addition, the _FTM- NN protein is labeled with Alexa Fluor 488 dye (Invitrogen (Carlsbad, CA); catalog number A20000) according to the manufacturer's instructions, followed by desalting and residual unbound Alexa Fluor. 488 dye was removed.

  Llama pre-immune PBMCs (background control) and immunized PBMCs were fluorescently labeled, IgGl (conventional heavy + light chain immunoglobulin), IgG2 and IgG3 (heavy chain immunoglobulin group) specific mouse monoclonal antibodies, fluorescently labeled Staining was performed using the DH59B antibody (CD172a) (VMRD, Inc. (Pullman, WA); catalog number DH59B; Davis et al. 1987, Vet. Immunol. Immunopathol. 15: 337-376) and Alexa 488 labeled antigen. TOPRO3 was included as a viable / dead cell discriminating dye. IgG1 + B lymphocytes, monocytes, neutrophils and dead cells were gated out and thereby excluded from sorting. Antigen-specific (A488 +) IgG2 or IgG3 positive B cells were single cells individually sorted into separate PCR plate wells containing RT-PCR buffer.

For llama 206, 1.9% of antigen-positive cells were obtained (1.0% in the pre-immune reference sample) of the total amount of live IgG2 / IgG3-positive cells, and for llama 207, 4.2% of positive cells were obtained. (0.7% in the pre-immune reference sample). The heavy chain variable region gene was directly amplified from these B cells by single-cell RT-PCR and nested PCR. The PCR product was subsequently cloned into a TOPO compatible expression vector derived from pUC119 containing the LacZ promoter, ampicillin or carbenicillin resistance gene, multiple cloning site and gen3 leader sequence. In frame with the Nanobody® coding sequence, the vector encoded a C-terminal c-myc tag and a (His) 6 tag. The cloned construct was transformed into TOP10 E. coli cells by high-throughput electroporation. Single clones were cultured in 96 deep well plates (1 ml volume) and Nanobody® expression was induced by adding IPTG. Periplasmic extracts (volume: ８０80 μl) were prepared by osmotic shock and analyzed for binding to _FTM- in a binding ELISA.

Briefly, 2 μg / ml _FTM- was directly immobilized on Maxisorp microtiter plates (Nunc). The released binding sites were blocked with 4% Marvel in PBS. Next, 10 μl of periplasmic extract containing Nanobodies® of different clones in 100 μl of 2% Marvel PBST was bound to the immobilized antigen. Following the incubation and washing steps, Nanobody® binding was revealed using a rabbit anti-VHH secondary antibody (for the periplasmic fraction). After the washing step, Nanobodies® in the periplasmic fraction were detected with an HRP-conjugated goat anti-rabbit antibody. The binding specificity was determined based on the OD value compared to a control not receiving Nanobody®.

A total of eight positive _FTM- NN binding factors (4 from llama 206 and 4 from llama 207) were obtained from the 52 cloned VHHs.

Example 5: Screening of Nanobodies® that bind to antigenic sites II or IV-VI Periplasmic extracts containing a single Nanobody® were subjected to an Alphascreen® assay (Perkin Elmer (Waltham, MA )) Was used to analyze for binding to antigenic sites II or IV-VI (Garcia-Barreno et al. 1989, J. Virol. 63: 925-932, Lopeze et al. 1998, J. Virol. 72: 6922). -6928). In this setting, _FTM- NN binds to Synagis® and the Fab of 101F simultaneously, allowing detection of Nanobodies® that prevent binding of each of the respective antigenic sites II and IV-VI. Here, the addition of periplasmic extracts _{F TM-} NN protein (0.3 nM), and incubated for 15 min. Subsequently, biotinylated Fab Synagis® (0.3 nM) and Fab 101F binding acceptor beads (10 μg / ml) were added and the mixture was incubated for 1 hour. Finally, streptavidin-coated donor beads (10 μg / ml) were added, and after 1 hour incubation, the plates were read on an Envision microplate reader. The periplasmic extract was diluted 25-fold (corresponding to a final concentration of approximately 40 nM). The assay was demonstrated by titration of the known competitors of Synagis®, mab 18B2 (Argene (Varilhes, France); 18042 N1902) and 2F7 (Abeam (Cambridge, UK); ab43812). Synagis® Fab, Numax Fab and 101F Fab were also classified as Numax Fab with the lowest IC50 value (8.6 × 10 ⁻¹¹ M), followed by Synagis® Fab (5.97 × 10 ⁻ M). ¹⁰ M) and 101F Fab (1.12 × 10 ⁻⁹ M). For screening of periplasmic extracts (25-fold dilution), both Numax Fab (40 nM) and 101F Fab (40 nM) were used as positive controls, while irrelevant periplasmic extracts were used as negative controls. Alphascreen (TM) in _{F TM-} NN binding of the protein as compared to the negative control hinder than 75% clones were identified as hits (hit). A total of 341 hits were identified from 1856 clones from all six llamas, but the majority were from llamas 206 and 207. In addition, three clones showed competition among eight clones obtained by nanoclone selection.

The exact antigenic site competition factor (II or _{IV~VI), F TM-} NN protein (1 [mu] g / ml) biotinylated for binding to Synagis (R) Fab (2 nM) or biotinylated 101F Fab and (3 nM) Analyzed by the competition ELISA used. Briefly, _FTM- NN protein was directly immobilized on Maxisorp microtiter plates (Nunc) and the released binding sites were blocked with 4% Marvel in PBS. The periplasmic extract was diluted 10-fold and mixed with the biotinylated Fab before binding to the immobilized _FTM- NN protein. Control periplasmic fractions selected for other viral coating proteins were included. Competing antibodies were bound to the immobilized antigen in the presence or absence of Nanobody®. After the incubation and washing steps, detection was performed with an extravidin-HRP conjugated secondary antibody (Sigma-Aldrich, St. Louis, MO, catalog number E2886). The binding specificity was determined based on the OD value compared to a control not receiving Nanobody®.

  All hits were subjected to sequence analysis and were classified into families according to their CDR3 sequences (June 5, 2009 entitled "Amino acid sequences directed against envelope proteins of avirus and polypeptides comprising the same for the treatment of viraldiseases"). (See Table C-4 and Table A-1 of International Application PCT / EP2009 / 056975, filed by Ablynx NV in U.S.A.).

Example 6: Screening of RSV Neutralizing Nanobodies® From all six hRSV libraries, 163 unique sequences (160 from the phage library and three from the nanoclone) were partially The protein was analyzed for its ability to neutralize RSV Long in a microneutralization assay as a purified protein. Hep2 cells were plated in 96-well plates at a density of 1.5 × 10 ⁴ cells / well in DMEM medium containing 10% fetal calf serum (FCS) supplemented with penicillin and streptomycin (100 U / ml and 100 μg / ml, respectively). And incubated at 37 ° C. for 24 hours in a 5% CO ₂ atmosphere. The virus stocks used were the virus stocks derived from ATCC VR-26, designated as hRSV strains long, Long LM-2 and Long M2 (used interchangeably) and whose F protein sequence corresponds to P12568 or M22643. is there. The virus stock was passaged several times from the ATCC stock. The sequence of the F protein was confirmed to be identical to P12568 (see Example 9). A standard amount of the hRSV strain Long LM-2 was pre-incubated with a fixed volume of 50 μl of a fixed volume of a serial dilution of purified Phytips Nanobody® (20 μl) at 37 ° C. for 30 minutes. The medium of Hep2 cells was replaced with a premix, and after 2 hours of infection, 0.1 ml of assay medium was added. Assays were performed in DMEM medium supplemented with 2.5% fetal calf serum and penicillin and streptomycin (100 U / ml and 100 μg / ml, respectively). After incubating the cells for an additional 72 hours at 37 ° C. in a 5% CO 2 atmosphere, the cells are washed twice with 0.05% Tween-20 in PBS and once with PBS alone, after which the cells are washed for 20 minutes at 4 ° C. , Fixed in 80% cold acetone (Sigma-Aldrich, St. Louis, MO) in PBS (100 μl / well) and dried completely. The presence of F protein on the cell surface was then detected in an ELISA type assay. In addition, the fixed Hep2 cells were blocked with 2% bovine serum albumin (BSA) in PBS for 1 hour at room temperature (than) and then incubated with Synagis® (2 μg / ml) for 1 hour. . For detection of goat anti-human IgG, Fcγ fragment-specific HRP (Jackson ImmunoResearch, West Grove, PA) was used, followed by ELISA developed according to standard procedures.

  As a positive control, in addition to the previously identified RSV neutralizing Nanobodies® 191D3 (SEQ ID NO: 9) and 192C4 (SEQ ID NO: 11) included in the screen, 5 antigenic site II clones in the strong RSV Long Showed sum activity: 1E4 (also referred to as 207D1; SEQ ID NO: 1), 7B2 (SEQ ID NO: 2), NC23 (SEQ ID NO: 3), and two members of the same family 15H8 (SEQ ID NO: 4) and NC41 (SEQ ID NO: 4). No. 5) (Table A-1). All of the antigenic sites IV-VI specific Nanobodies® showed very weak neutralizing activity against the hRSV Long LM-2 strain.

Example 7: Generation of hRSV Nanobodies® In addition to the previously identified RSV neutralizing Nanobodies® 191D3 (SEQ ID NO: 9) and 191E4 (SEQ ID NO: 10) included in the screen as a positive control, Five new neutralizing Nanobodies® (1E4, 7B2, 15H8, NC23 and NC41) selected from the above screen, and one antigenic site IV-VI Nanobodies® (15B3, SEQ ID NO: 7) ) Was expressed, purified and further characterized. In addition, the coding sequence was recloned into an expression vector derived from pUC119 containing the LacZ promoter, kanamycin resistance gene, multiple cloning site and OmpA signal peptide sequence. In frame with the Nanobody® coding sequence, the vector encoded a C-terminal c-myc tag and a (His) 6 tag.

  Expression was performed in E. coli TG-1 cells as c-myc, His6-tagged protein in a 1 L culture volume. Expression was induced by adding 1 mM IPTG and continued at 37 ° C. for 3 hours. After spinning the cell culture, the periplasmic extract was prepared by freeze-thawing the pellet and resuspending it in dPBS. These extracts were used as starting material for immobilized metal affinity chromatography (IMAC) using a Histrap FF crude column (GE healthcare (Uppsala, Sweden)). Nanobodies® were eluted from the column with 250 mM imidazole, followed by desalting to dPBS.

Example 8: hRSV Nanobodies binding all purified Nanobodies of the F protein in characterization ELISA (R) _were shown to bind to F protein in binding ELISA against _{F TM-} NN proteins and hRSV . The results of hRSV binding are shown in Table B-1. Briefly, 1 [mu] g / ml of _{F TM-} NN or 5 [mu] g / ml of hRSV (Hytest (Turku, Finland) ) were immobilized directly Maxisorp microtiter plates. The released binding sites were blocked with 1% casein. Serial dilutions of purified Nanobody® were allowed to bind to the antigen for 1 hour. Final detection with rabbit anti-VHH secondary antibody and HRP-conjugated goat anti-rabbit antibody was used to demonstrate Nanobody® binding. Binding specificity was determined based on the OD value compared to an irrelevant Nanobody® control.

Kinetic analysis was performed using surface plasmon resonance analysis on the _FTM- NN protein to determine the correct binding affinity of the purified Nanobody®. For the Preincubation of Sensorchip CM5, 10 μg / ml hRSV F _TM- protein was left for 120 seconds. For immobilization by amine coupling, EDC / NHS was used for activation and ethanolamine HCl was used for deactivation (Biacore, amine coupling kit). 100 nM Synagis® and then 100 nM Nanobody® were added. Evaluation of off-rates was performed by fitting a 1: 1 interaction model (Langmuir binding model) with Biacore's T100 software v1.1. The dissociation rate and affinity constant are shown in Table B-1.

Competition with Synagis (R) The ability of purified Nanobodies (R) to compete with Synagis (R) Mab or Biotinylated Synagis (R) Fab for binding to _FTM- NN was performed in a competitive ELISA Determined according to the procedure essentially as described in Example 5. FIG. 1 shows a representative example of a competitive ELISA, where purified Nanobodies® compete with Biotinylated Synagis® Fab for binding to _FTM- NN. The E50 values are summarized in Table B-1.

Binding to F protein in Biacore Example 9: In vitro microneutralization of separate hRSV strains The efficacy of purified Nanobodies® in neutralizing various A and B type RSV strains was determined by an in vitro microneutralization assay. (See Example 6). Virus stocks of RSV Long LM-2 (Accession No. P12568; ATCC VR-26), RSV A-2 (ATCC VR-1540; Lot No. 31998840) and RSV B-1 (ATCC VR-1580; Lot No. 5271356) were used. , Prepared in Hep2 cells, and subsequently titrated to determine the optimal infection dose to use in the microneutralization assay. The results of the neutralization efficacy of various purified Nanobodies® are shown in Table B-1. All six Nanobodies® recognizing the SYNAGIS® epitope were able to efficiently neutralize type A Long and A-2 strains, but neutralized infection by strain B-1 Could not be done or neutralized infection at concentrations above 1 μM. The 101F competitors 15B3 and 191E4 showed very weak neutralizing potency against strain B-1 only when administered at μM concentration.

  The sequence of each F protein of the various RSV strains was verified by reverse transcriptase PCR and subsequent sequence analysis. Briefly, total RNA was isolated from RSV-infected Hep2 cells using the RNeasy mini kit (Qiagen (Venlo, Netherlands)) and then complemented using the Superscript III reverse transcriptase kit (Invitrogen (Carlsbad, CA)). DNA was prepared. The F protein of the RSV A strain was amplified and sequenced using primers described in Kimura et al. 2004 (Antiviral Research 61: 165-171). For the amplification of the F protein of the RSV B-1 strain, the following primers were used: FB1_outer_for: cttagcagaaaaccgtga (SEQ ID NO: 13); FB1_outer_rev: tgggttgatttgggattg (SEQ ID NO: 14); -Rev: gctgacttcacttggtaa (SEQ ID NO: 16). The sequence of the RSV B-1 strain corresponded to accession number P13843 (including an additional point mutation Ser540Leu). The sequence of the RSV Long M2 strain completely corresponded to the reported sequence (accession number M22643). The sequence of the RSV A-2 strain corresponded to accession number M11486. See also Table A-2.

Example 10: Construction, production and characterization of polyvalent hRSV Nanobodies® Polyvalent Nanobodies® constructs linked by Gly-Ser linkers of different lengths and compositions were combined with different primers containing specific restriction sites Separate PCR reactions using the set (one N-terminal, one intermediate (if trivalent), one C-terminal to the Nanobody® subunit) were generated. Similarly, a multivalent construct linked by an Ala-Ala-Ala linker was generated. All constructs were cloned into an expression vector derived from pUC119 containing the LacZ promoter, kanamycin resistance gene, multiple cloning site and OmpA signal peptide sequence. In frame with the Nanobody® coding sequence, the vector encoded a C-terminal c-myc tag and a (His) 6 tag. When a 35Gly-Ser linker was present in the multivalent construct, an expression vector derived from pUC119 containing the LacZ promoter, kanamycin resistance gene and OmpA signal peptide sequence was used. Directly downstream of the signal peptide, a multiple cloning site for Nanobody® insertion is followed by a DNA sequence encoding a 35Gly-Ser linker, and for cloning of a second Nanobody® sequence. A second multicloning site was present. In frame with the resulting Nanobody®-35Gly-Ser-Nanobody® coding sequence, the vector encoded a C-terminal c-myc tag and a (His) 6 tag. The multivalent constructs generated using RSV-specific Nanobodies® are listed in Table B-2. The sequence of the multivalent construct is shown in Table A-3.

  The multivalent RSV Nanobody® construct was expressed, purified, and further characterized. Production was performed in E. coli TG1 cells, followed by purification via a His tag from the periplasmic fraction by IMAC and desalting essentially as described in Example 7. Certain trivalent constructs (eg, RSV401, RSV404, RSV406) were made in P. pastoris and subsequently purified from media fractions. All trivalent Nanobodies® were subjected to gel filtration as a final step to remove possible divalent and monovalent degradation products.

Purification multivalent Nanobodies (TM) and the binding of hRSV F _protein, (see Example 8) was confirmed in ELISA against both _{F TM-} proteins and hRSV. For the majority of Nanobodies®, formatting to divalent and trivalent constructs showed similar EC50 values as their monovalent counterparts (as shown in FIG. 2 7B2) 7B2 and Except for the NC23 multivalents, it produced a clear but limited (up to 10-fold increase) avidity effect.

Example 11: Efficacy of bivalent and trivalent constructs to neutralize hRSV The efficacy of Nanobody® constructs was evaluated in RSV neutralization assays against various RSV strains (see Examples 6 and 9). Want). Bivalent Nanobodies® that bind to antigenic site II show a significant increase (100- to 1000-fold) in potency in neutralizing Long compared to their monovalent counterparts (ie, IC50 values ranged from 50 pM to 380 pM (better than or similar to Numax Fab). However, for the RSV B-1 strain, the increase in potency was not as strong, and none of the dimeric constructs was more potent than SYNAGIS®. Surprisingly, as shown in FIG. 3, this could be overcome by generating a trivalent construct. The trivalent construct with three Nanobodies® binding to antigen site II was a neutralizer at least 1000-fold more potent against its RSV B-1 strain than its monovalent counterpart.

Example 12: Reactivity of monovalent Nanobodies® with escape mutants of Long strain
Antigen site II (R47F / 4, R47F / 7, RAK13 / 4, R7C2 / 11, R7C2 / 1) or antigen site IV described in Lopez et al. 1998 (J. Virol. 72: 6922-6928). A number of escape mutants specific for VI (R7.936 / 1, R7.936 / 4, R7.936 / 6, R7.432 / 1), or a combination of both (RRA3), were identified as antigen site II Or selected to test its reactivity with ten monovalent Nanobodies®, including Nanobodies® 191C7 (SEQ ID NO: 8) previously identified as not binding to antigenic sites IV-VI. did.

  This assay was performed according to Lopez et al. 1998 (J. Virol. 72: 6922-6928). Briefly, each Nanobody® was tested at 0.2 μg / ml in an ELISA using antigen extracts of HEp-2 cells infected with various escape mutants. The absorbance results were normalized for reactivity against the reference virus (Long wild type) strain and the control Nanobody® 191C7. The results are shown in Table B-3.

  Reactivity greater than 75% is shown as solid black cells, darkly shaded cells correspond to 75% -50% reactivity, and lightly shaded cells correspond to 25% -50% reactivity. , Less than 25% reactivity is indicated by blank cells. In general, Nanobodies® (192C4, 191D3, 191F2, NC23, 15H8, 7B2, and NC41), previously identified as antigen site II binding agents in the previous examples, were modified for typical mutations in antigen site II. Other Nanobodies®, previously identified as antigenic sites IV-VI binding factors, were in fact sensitive to mutations at these sites.

Example 13: Reactivity of polyvalent Nanobodies® with escape mutants of the Long strain A large number of multivalent constructs were subsequently analyzed against a limited population of escape viruses to evaluate binding. This assay was performed according to Lopez et al. 1998 (J. Virol. 72: 6922-6928). Briefly, each Nanobody® was compared to 0.1 μg for monovalent Nanobody® in an ELISA using antigen extracts of HEp-2 cells infected with various escape mutants. / Ml, divalent and trivalent Nanobodies® were tested at 0.05 μg / ml. The absorbance results were normalized for reactivity against a reference virus (Long wild type) strain and a control Nanobody® (191E4 in this particular assay; SEQ ID NO: 10). The results are shown in Table B-4.

  Reactivity greater than 75% is shown as solid black cells, darkly shaded cells correspond to 75% -50% reactivity, and lightly shaded cells correspond to 25% -50% reactivity. , Less than 25% reactivity is indicated by blank cells. Notably, the multivalent construct showed improved binding to the mutant virus R7C2 / 11 compared to its monovalent counterpart. Also, the dual paratopic construct RSV403 was not sensitive to any of the mutants.

Example 14: Neutralization of Escape Mutants in Long Strain by Multivalent Nanobodies® In Examples 12 and 13, typical antigenic sites II and / or IV-VI RSV escape mutants The binding of monovalent Nanobodies® to was described. The binding of Nanobodies® specifically recognizing these antigenic sites was almost lost or significantly reduced. Formatting of these Nanobodies® into bivalent or trivalent constructs partially restored binding activity (but not for all three escape mutant viruses). Binding to the escape mutant R7C2 / 1 (mutation K272E at antigen site II) is 25% for any divalent or trivalent construct consisting solely of Nanobodies® binding to antigen site II. Remained below the level. Nanobodies® 15B3 and 191E4, which bind to antigenic sites IV-VI, are the only ones that can bind to this mutant (by itself or in a dual paratopic construct) at a level of 75% or more. Nanobody (registered trademark).

  A more detailed analysis of the data indicated that binding to R7C2 / 1 increased slightly with increasing Nanobody® valency. The binding of the 7B2 construct was 0%, 4.4% and 13% for monovalent, bivalent (RSV106) and trivalent (RSV400) formats, respectively. Such low levels of residue binding are expected to result in a very large loss of RSV neutralizing potency.

  The neutralizing potency of Nanobody® was evaluated against the same selected set of escape mutants as described in Example 13. For this purpose, the monovalent Nanobodies® 7B2, 15H8 and NC41 were compared with their respective trivalent counterparts RSV400, RSV404 and RSV407. Note that in Example 13, only RSV400 was evaluated for binding to these escape mutants. The double paratopic trivalent molecule RSV403 (7B2-15B3-7B2) was also analyzed for its neutralizing ability.

The hRSV microneutralization assay was performed essentially as described in Example 6. Briefly, Hep2 cells were placed in a 96-well plate at a concentration of 1.5 × 10 ⁴ cells / well in 10% fetal calf serum (FCS) supplemented with penicillin and streptomycin (100 U / ml and 100 μg / ml, respectively). And incubated for 24 hours at 37 ° C. in a 5% CO ₂ atmosphere. Virus stocks of various viruses were prepared in Hep2 cells and subsequently titrated to determine the optimal infection dose for use in the microneutralization assay. A standard amount of the specific hRSV strain was pre-incubated with a serial dilution of purified Nanobody® in a total volume of 50 μl at 37 ° C. for 30 minutes. The medium of Hep2 cells was replaced with the premix and infected for 2 hours, after which 0.1 ml of assay medium was added. Assays were performed in DMEM medium supplemented with 2.5% fetal calf serum and penicillin and streptomycin (100 U / ml and 100 μg / ml, respectively). After incubating the cells for an additional 72 hours at 37 ° C. in a 5% CO 2 atmosphere at 37 ° C., the cells are washed twice with 0.05% Tween-20 in PBS and once with PBS alone before the cells are washed in PBS. With 100% cold acetone (Sigma-Aldrich (St. Louis, MO)) (100 μl / well) at 4 ° C. for 20 minutes and dried completely. Next, the presence of the F protein on the cell surface was detected in an ELISA type assay. In addition, the fixed Hep2 cells were blocked with 5% porcine serum albumin in PBS for 1 hour at room temperature, and then anti-F protein polyclonal rabbit serum (Corral et al. 2007, BMC Biotechnol. 7:17) or Incubated with SYNAGIS® (2 μg / ml) for 1 hour. For detection of goat anti-rabbit HRP-conjugated antibodies or goat anti-human IgG, Fcγ fragment-specific HRP (Jackson ImmunoResearch, West Grove, PA) was used, followed by ELISA developed according to standard procedures.

  As shown in FIGS. 4A-4C, monovalent Nanobodies® had little ability to neutralize antigenic site II escape mutant viruses R7C2 / 11 and R7C2 / 1. The potency to neutralize the R7.936 / 4 antigenic site IV-VI mutant was comparable to the potency to neutralize the wild-type Long strain. These data are consistent with the binding data of Example 12 and the epitope mapping described for these Nanobodies® in Example 5.

  However, the Trivalent Nanobody® construct strongly neutralized all three escape mutants (FIGS. 4D-4G). Although maximum inhibition was observed at concentrations as low as about 20 nM, this level of inhibition was not observed for monovalent Nanobodies at concentrations up to 2 μM. Strong neutralization of R7C2 / 1 (approximately equal to neutralization of R7C2 / 11), but a very large loss of neutralizing potency in Example 13 is expected to result in a very large loss of neutralizing potency. The most surprising is that significant losses were shown. At a concentration of about 0.2 μM, the bivalent IgG Synagis® Palivizumab, which also recognizes antigenic site II, could hardly block R7C2 / 1 or R7C2 / 11 replication. At this concentration, the IC50 was not reached and R7.936 / 4 and wild-type Long virus were neutralized with a few nM IC50 (data not shown).

Example 15: Analysis of the effect of linker length on the potency of NC41 trivalents To determine the effect of linker length of the NC41 trivalent, range from 3Ala linker, 9GS linker, 15GS linker to 20GS linker. Constructs (RSV408, RSV409, RSV407 and RSV410, respectively) having the following linkers were generated. All four NC41 trivalents were able to completely neutralize both RSV B-1 and Long strains (FIG. 5). In neutralizing the RSV Long strain, no effect of linker length was observed because all constructs were equally effective. In contrast, the IC50 values for the B-1 strain of the construct with the 9GS linker and the 3Ala linker were increased, indicating that a minimum linker length of 15GS was required for maximum efficacy. This indicates that the divalent NC41 construct is already a very potent neutralizing agent for the Long strain, but the potency difference between the divalent and trivalent NC41 for the B-1 strain is much greater. This can be explained by the observation that it is large (see Example 11). With RSV 408 and RSV 409, the accessibility of the intermediate Nanobodies may not be very optimal.

Example 16: Humanization of Nanobody® NC41 The sequence of Nanobody® NC41 was converted to human germline VH3- so that residues suitable for further humanization of the Nanobody® sequence could be selected. 23 and aligned. In addition, computer analysis was performed to identify residues that were likely to be post-translationally modified (eg, Asp isomerization) and identified mutations that could improve chemical stability. CDR regions and so-called characteristic residues known to be essential for the stability and efficacy of Nanobodies® have been excluded for modification.

  For NC41, a total of 11 positions were selected for mutation to the corresponding human residue. The four mutations (Val5Leu, Ala14Pro, Glu44Gly, Gln108Leu) indicate that these residues can dramatically affect Nanobody® function (based on data from other Nanobodies®). Since it was not expected, it was introduced at the same time. In this basic mutant, seven residues that are unknown whether mutations to the human counterpart are tolerated are mutated using a library approach (Ser19Arg, Ile20leu, Ala74Ser, Gly78Leu, Ala83Arg). , Asp85Glu, Arg105Gln) at each position, either wild-type amino acids or the corresponding human amino acids. The resulting library with a theoretical diversity of 128 was generated by gene assembly using overlapping oligonucleotide sequences containing degenerate codon usage, followed by the LacZ promoter, kanamycin resistance gene, multicloning The site was cloned into an expression vector derived from pUC119 containing the OmpA leader sequence. In frame with the Nanobody® coding sequence, the vector encoded a C-terminal c-myc tag and a (His) 6 tag. Nanobodies® were made in the periplasm of E. coli (see Example 7). Library diversity was confirmed by sequence analysis.

  Periplasmic extracts from 368 individual NC41 mutants and wild-type NC41 were generated and subjected to a functional screening cascade to identify the best humanized NC41 mutants in terms of both potency and stability. In the first step, RSV binding between the humanized NC41 mutant and the RSV Long strain was determined in an ELISA (Hytest, Turku Finland; # 8 RSV79) (see Example 8).

  In addition, positive binding agents were analyzed for binding to Hep2 cells infected with RSV B-1 strain. Here, Hep2 cells were seeded in 96-well plates essentially according to the procedure described for the neutralization assay and infected with the RSV B-1 strain (see Example 6). Three days later, cells were fixed with ice-cold acetone and plates were used in ELISA assays using various dilutions of periplasmic extract. Nanobodies® binding to Hep2-B1 infected cells were detected using an anti-VHH rabbit polyclonal antibody followed by a goat anti-rabbit HRP conjugated antibody, followed by ELISA developed according to standard procedures.

  In addition, periplasmic extracts of all binding factors were heated for 2 hours to 74 ° C., 5 ° C. above the melting temperature of wild-type NC41, to verify whether the introduced mutations affected temperature stability. . Binding to the RSV long strain before and after heating was analyzed by ELISA, and the ratio of the binding signal after heating to before heating was used as a measure for temperature stability.

Finally, the kinetic dissociation rate of the mutant was determined in a Biacore assay for the _Ftm- NN protein as described in Example 8.

  All binding agents were sequenced and ranked by their ability to bind to the RSV F protein. When analyzing the sequence of the strongest binding factor, a clear preference for Gln105 (human residue) was observed in all cases. The Ile20Leu mutation was considered underrepresented, but for all other positions there was no apparent preference for either wild-type or human sequences, and the mutant was compared to wild-type NC41. It contained up to 10 mutations. Notably, an additional point mutation (Gly54Asp) was observed in the CDR2 region in one mutant. This mutant (NC41 mutant 6) showed the lowest off-rate of all mutant and wild-type NC41, resulting in increased affinity.

  Based on the sequence and functional data, 18 variants (Table A-4) were selected for further characterization as a purified protein (Figures 6 and 7). All mutants were made and purified, and the potency for neutralizing RSV Long and B-1 strains was determined in a microneutralization assay. Most mutants showed very similar activity to wild-type NC41, but some mutants showed increased potency against both Long (2 fold) and B-1 (6 fold) strains The strongest neutralizers were NC41 mutants 6, 8, 9, 17 and 18. Of note, mutant 18 was maximally humanized at all 11 positions, with additional Asp54 introduced into the CDR2 region. Mutants 10 and 11 were more potent than NC41 in neutralizing the B-1 strain, but not against the Long strain.

For a selection panel of NC41 variants, kinetic binding parameters were determined in Biacore for the _Ftm- NN protein as described in Example 8 (Table B-5). In the calculated data, no significant difference was observed between NC41 and humanized NC41 mutants 6, 8, and 17. Note that the binding rates of all NC41 variants were at the detection limit of the instrument, but the dissociation rates could be ranked as v06 <v17 <NC41 <v08. The effect of the Gly to Asp mutation (position 54) in CDR2 is clearly demonstrated when v17 and v18 are compared, as this mutation is the only difference in these maximally humanized variants. I was able to. As shown in Table B-5, neutralization was tested for both the Long and B-1 strains in two independent assays compared to the NC41 wild type. NC41v18 was more potent than NC41 against both viruses in both assays, and NC41v18 was more potent than NC41v17 against the Long strain in both assays. Also, improved neutralization of NC41v18 was observed for the B-1 strain in the second assay.

  All NC41 variants were subjected to heat-induced unfolding to evaluate the effect of the introduced mutation on protein stability. In addition, the melting temperature (Tm) was determined by the stepwise increase in temperature in the presence of Sypro Orange (a dye that binds to Trp residues exposed by unfolding of the protein). All mutants were shown to have an increased Tm (up to 9 ° C for mutant 18) compared to wild-type NC41 (69 ° C).

Example 17: Further sequence optimization for expression The sequence of Nanobody® NC41 was further analyzed for the purpose of optimizing expression in Pichia pastoris. NC41 variants 19-26 were designed by combining humanized positions that were found to be tolerated without loss of potency in the microneutralization assay (Example 16). Four mutations were introduced simultaneously (Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu) and three mutations were made both individually and in each possible test combination (Ala83Arg, Asp85Glu, Arg105Gln) (Table A-4). Please see). After cloning all constructs into expression vectors and introducing them into Pichia pastoris XL-33 strain, the number of integrations in the Pichia genome was evaluated by quantitative real-time PCR. From each construct, one clone with one to two copies and one clone with four or more copies were selected for small-scale production and the expression yield of the NC41 mutant relative to wild type was determined by SDS. -Inferred by gel electrophoresis on a PAGE gel. The NC41 mutants 20, 22, 24, 25 and 26 showed the highest expression, with low copy numbers and already expression levels comparable to wild-type NC41.

  The NC41 mutants 22 and 26 were recloned into an E. coli expression vector for production and purification of Myc-His tagged Nanobodies (see Example 7). The potency of both mutants for neutralizing RSV Long was tested in a microneutralization assay as described in Example 6. Both mutants were equal in potency to wild-type NC41 (IC50 of 126 nM and 310 nM for v22 and v26, respectively).

Example 18: Optimization of the sequence of RSV407 for chemical stability During production of Nanobodies and polypeptides of the invention, pyroglutamic acid (pGlu) was observed (by RP-HPLC) at the amino terminus. After the fermentation, more than 15% pGlu levels were detected, and during the stability test, the pGlu levels gradually increased during storage. In this way, the N-terminal glutamic acid (E) was converted to aspartic acid (D). This eliminated the possibility of N-terminal pGlu post-translational modification and increased the stability of the product.

  The amino acid sequences of the sequence-optimized Nanobody® variants are each given in Table A-5 (RSV434, SEQ ID NO: 142). For the production of Nanobody (registered trademark), a Pichia expression system was developed based on a commercially available system manufactured by Invitrogen using X-33 as a host strain. This system utilizes the AOX1 promoter to drive the production of Nanobodies® and uses α-conjugated secretory peptides to secrete Nanobodies® into the medium.

  The expression level of sequence optimized RSV434 compared to the parent molecule (RSV407) for RSV neutralization was analyzed by RP-HPLC, SE-HPLC.

  There was no significant difference in expression levels compared to RSV407, with both low and high copy number clones yielding> 1 g / L of clarified medium (without cells).

  A 1 ml sample was captured on a 1 ml MEP Hypercel column, eluted and analyzed by SE-HPLC and RP-HPLC. SE-HPLC analysis showed monomeric material and, as expected, RP-HPLC analysis clearly showed the absence of the pGlu post peak.

  RSV434 was further purified from the medium, captured on a MEP HyperCell column, and purified by anion exchange chromatography.

  The hRSV microneutralization assay was performed essentially as described in Example 6. FIG. 9 shows neutralization against both RSV Long and B-1 strains by both RSV407 and its sequence optimized variant RSV434.

Example 19: Preparation of a humanized and / or sequence-optimized multivalent construct of NC41 Nanobody® The NC41 humanized variant of Example 16 was prepared using a 15GS linker (sequences are shown in Table A-5). Formatted as a trivalent construct. Trivalent was prepared and purified as described in Example 10. FIG. 8A shows the neutralization by two of the trivalent humanized NC41 variants and their corresponding monovalent Nanobodies® against both RSV Long and B-1 strains. FIG. 8B shows the neutralization of the trivalent NC41 mutant against both RSV Long and B-1 strains. The IC50 values for neutralization of the RSV Long strain and the B-1 strain by the trivalent NC41 mutant are shown in Table B-7. Similar to the parent NC41 trivalent (RSV407), the humanized NC41 mutant trivalent was a Long neutralizer approximately 60-fold more potent than SYNAGIS®. In strain B-1, the trivalent is a more potent neutralizer than Synagis, and here slightly improved in order of RSV427>RSV426> RSV414 compared to the parent NC41 RSV407 trivalent. Thus, it was thought that the increased potency of the monovalent mutant against B-1 resulted in a slight improvement of the trivalent.

  A number of additional trivalent constructs (Table B-6) were generated based on the further sequence optimization shown in Examples 17 and 18. All constructs were cloned into Pichia pastoris expression vectors, transformed into Pichia, fermented, and tested for expression levels, stability and potency. RSV440 (variant 26) and RSV441 (variant 22) showed high expression levels in both small shake flask expression and fermentation.

Example 20: In Vitro Efficacy of Multivalent Constructs The ability to neutralize Nanobodies RSV434 and Synagis was evaluated in a plaque reduction assay against 31 RSV / A clinical isolates and 30 RSV / B clinical isolates. Both anti-RSV compounds and Synagis were tested at a single concentration of 40 μg / ml.

  Both Synagis and RSV434 effectively resulted in at least a 100-fold reduction in virus titer compared to the PBS control in 87% and 97% of the strains, respectively (Table B-8). In addition, RSV434 showed a greater neutralizing ability as compared to Synagis. The majority of RSV strains (84%) were completely inhibited by RSV434, and significantly less strains (20%) were completely inhibited by Synagis (Table B-8).

Example 21 In Vivo Efficacy of Multivalent Constructs The cotton rat model is a golden standard model with RSV. In this model, cotton rats were infected with the RSV / Tracy strain (day 0) and four days after infection virus titers and viral RNA were evaluated in lung lavage and nasal lavage. In the prophylactic configuration, a significant and dose-dependent reduction in viral load was observed for intranasal administration of RSV407 24 hours prior to RSV infection (FIG. 10).

  In addition, infected cotton rats were treated with Nanobodies at 24 or 48 hours post-infection, thereby investigating a therapeutic approach that mimics the situation in RSV-infected humans. In both cases, significant inhibition of virus replication was observed (Table B-9 and FIG. 11).

  In all experiments performed to date, Nanobodies were delivered intranasally as a mimic of pulmonary delivery. Using this route of administration, 9% to 41% of the Nanobodies were available in the lung as assessed by ELISA with bronchial lavage fluid collected immediately after administration.

Virus detection was delayed until day 7 post inoculation to avoid possible interference of residual Nanobodies in lung lavage fluid. Separate pharmacokinetic studies performed on Sprague Dawley rats estimated the half-life of Nanobodies in the lung to be approximately 10.8. On day 7, 5-6 days after the last dose, the Nanobodies are sufficiently removed from the lungs until there is no interference in the assay. Viral load in the positive control animals was reduced from ¹⁰ 5 pfu / ml to ¹⁰ 2 pfu / ml. Nevertheless, it was still possible to show a log reduction of about 0.6 (about 0.6 log) by treatment with RSV407 (Table B-9).

  As an alternative approach, quantitative PCR was developed to detect viral RNA. In both prophylactic and therapeutic experiments, a significant reduction in RSV RNA was observed (Table B-9).

Example 22: Generation of RSV escape mutant RSV Long escape mutant after culturing RSV Long in the presence of Nanobody near the respective IC90 concentration to identify critical contact residues with F protein Was analyzed. To examine whether both monovalent NC41 (5 μg / ml) and its trivalent RSV407 (2.5 ng / ml), as well as constructs recognizing two different epitopes, affect the time frame of viral escape occurrence Bispecific trivalent RSV413 (NC41-15B3-NC41) was used. After 12 consecutive incubations of Long in Hep2 cells in the presence of Nanobodies, virus elongation was observed under conditions with monovalent NC41, but surprisingly under conditions with trivalent Nanobodies. Did not. After repeated purification of a single viral stock from plaques, the sequence of the potential escape mutant F protein could be determined. Two different escape mutants were identified: NC41, NC41 / 13 with the mutation of N262Y, and NC41 / 17 containing the mutation N276Y.

Table A-1: Sequence of monovalent Nanobodies® binding to RSV F protein

Table A-2: Sequence of F protein

Table A-3: Amino acid sequences of multivalent constructs that bind to hRSV

Table A-4: Sequence of humanized and / or sequence optimized NC41 variants

Table A-5: Amino acid sequences of multivalent humanized and / or sequence-optimized constructs that bind hRSV

Table A-6: Preferred combinations of CDR sequences

Table A-7: Shown below the linker sequence:
Linker sequence number sequence hinge

Table B-1: Characterization of Nanobodies® binding to hRSV F protein Show below:
Clone Family Epitope Binding Competitive Synagis®
Reaction rate analysis RSV neutralization

Table B-2: Nomenclature of multivalent Nanobody® constructs directed to hRSV F protein Show below:
Type name construct SEQ ID NO bivalent double paratope trivalent

Table B-3: Reactivity of monovalent Nanobodies® with antigen extracts of HEp-2 cells infected with various escape mutants of the Long strain Show below:
Virus Nanobody (registered trademark)
Amino acid substitution

Table B-4: Reactivity of monovalent and bivalent Nanobodies® with antigen extracts of HEp-2 cells infected with various escape mutants of the Long strain Show below:
Virus Nanobody (registered trademark)
Amino acid substitution

Table B-5: Neutralization and kinetic binding parameters in Biacore for _Ftm- NN protein for selected NC41 variants Show below:
Name Neutralized Biacore Synergy

Table B-6: Trivalent humanized and / or sequence optimized variants of NC41 Show below:
Name construct sequence number

Table B-7: Neutralization of Long and B-1 by trivalent NC41 mutant Show below:
Experimental series of ratios for trivalent synergies

Table B-8: Neutralizing capacity of RSV434 and Synagis in plaque assays against various clinical isolates Shown below:
Number reduced by 100 times or more (%)
Comparison between test drugs Number of complete virus inhibition (%)
RSV group Synagis total P value

Table B-9: Schematic of all cotton rat experiments performed showing detection of both replicable virus and viral RNA 4 days after inoculation (experiment 3 also detected on day 7)
Show below:
Differences in viral load relative to Nanobody dose treatment plan controls Viable reduction of viral RNA Readout on day 7 of treatment day

FIG. 3A
Synagis Mab Synagis monoclonal antibody Figure 3B
Synagis 4A
% Viral antigen Viral antigen (%)
FIG. 4B
% Viral antigen Viral antigen (%)
FIG. 4C
% Viral antigen Viral antigen (%)
FIG. 4D
% Viral antigen Viral antigen (%)
FIG. 4E
% Viral antigen Viral antigen (%)
FIG. 4F
% Viral antigen Viral antigen (%)
FIG. 4G
% Viral antigen Viral antigen (%)
FIG.
Synagis Figure 6
# mutations Number of mutations Figure 8A
Synagis Figure 8B
% Viral antigen Viral antigen (%)
Synagis Figure 9A
A strain A strain figure 9B
B strain B strain figure 10A
Lung lavage virus titers Lung lavage virus titer
Log10 PFU / g lung Log10 PFU / g (lung)
TREATMENT processing figure 10B
Lung lavage q-RT-PCR q-RT-PCR of lung lavage
Reduction magnification FOLD REDUCTION IN MEAN C _T VALUES mean _{C T} value
TREATMENT processing figure 11A
MEAN (± SD) VIRUS TITER (log10 PFU / g lung) Average (± SD) virus titer (log10 PFU / g (lung))
v Untreated
TREATMENT (mean mg / kg) treatment (average (mg / kg))
FIG. 11B
MEAN (± SD) VIRUS TITER (log10 PFU / g lung) Average (± SD) virus titer (log10 PFU / g (lung))
v Untreated
TREATMENT (mean mg / kg) treatment (average (mg / kg))

Claims (19)

The amino acid residue is
Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Gly78Leu, Ala83Arg, Asp85Glu, and Arg105Gln,
Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54Asp,
Gly54Asp,
Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Ala83Arg,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Asp85Glu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Arg105Gln,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Asp85Glu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Arg105Gln,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Asp85Glu, and Arg105Gln, or Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, Asp85Glu, and Arg105Gln.
Of mutated, or at least one Nanobody consisting of the amino acid sequence of SEQ ID NO: 5 (R), or this or Ru Rana, polypeptide. Further comprising a polypeptide according to claim 1 one or more other amino acid binding units. 3. The polypeptide of claim 2 , further comprising one or more other amino acid binding units linked via one or more peptide linkers. The polypeptide according to any one of claims 1 to 3 , which is a multivalent polypeptide. Even without least including Nanobodies are three identical, multivalent polypeptide of claim 4. The amino acid residue is
Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Gly78Leu, Ala83Arg, Asp85Glu, and Arg105Gln,
Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54Asp,
Gly54Asp,
Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Ala83Arg,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Asp85Glu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Arg105Gln,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Asp85Glu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Arg105Gln,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Asp85Glu, and Arg105Gln, or Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, Asp85Glu, and Arg105Gln.
Of mutated, having SEQ ID NO: 5, or at least three Nanobodies (TM), or this or Ru Rana, multivalent polypeptide. The polyvalent polypeptide according to any one of claims 4 to 6 , which is divalent or trivalent. In at least one Nanobody® forming part of SEQ ID NO: 53, the amino acid residue is
Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Ala83Arg, Asp85Glu, Arg105Gln, and Gln108Leu,
Gly78Leu, Ala83Arg, Asp85Glu, and Arg105Gln,
Val5Leu, Ala14Pro, Glu44Gly, Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Ala83Arg, Asp85Glu, Arg105Gln, Gln108Leu, and Gly54Asp,
Gly78Leu, Ala83Arg, Asp85Glu, Arg105Gln, and Gly54Asp,
Gly54Asp,
Ala14Pro, Ser19Arg, Ile20Leu, and Gln108Leu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Ala83Arg,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Asp85Glu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, and Arg105Gln,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Asp85Glu,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, and Arg105Gln,
Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Asp85Glu, and Arg105Gln, or Ala14Pro, Ser19Arg, Ile20Leu, Gln108Leu, Ala83Arg, Asp85Glu, and Arg105Gln.
Underwent mutation, or comprising the amino acid sequence of SEQ ID NO: 53, or this or Ru Rana, trivalent polypeptide. Nucleic acid encoding the polypeptide de according to any one of claims 1-8. Expressing polypeptide de according to any one of claims 1-8, or may be expressed under suitable conditions; comprising a nucleic acid according to and / or claim 9, (excluding humans) host Or a host cell. Polypeptide according to any one of claims 1-8, or comprising a nucleic acid according to claim 9, composition. The composition according to claim 11 , which is a pharmaceutical composition. And polypeptides de according to any one of claims 1-8, and a carrier for pulmonary delivery, pharmaceutical compositions. Polypeptide according to any one of claims 1-8, or a method for producing a composition according to any one of claims 11 to 13,
In (excluding humans) host or host cell according to a) claim 10, step to express the nucleic acid according to claim 9, or of Claim 10 (excluding humans) host or host cell, wherein polypeptide according to any one of claims 1-8, or under conditions such that the expression and / or produce a composition according to any one of claims 11 to 13, wherein said host or said host A method comprising at least the step of culturing and / or maintaining cells. Polypeptide according to any one of claims 1-8, or a method for producing a composition according to any one of claims 11 to 13,
In (excluding humans) host or host cell according to a) claim 10, step to express the nucleic acid according to claim 9, or of Claim 10 (excluding humans) host or host cell, wherein polypeptide according to any one of claims 1-8, or under conditions such that the expression and / or produce a composition according to any one of claims 11 to 13, wherein said host or said host cells step of culturing and / or maintaining and subsequently b) obtained in this way, comprising at least the step of isolating and / or purifying the polypeptides de according to any one of claims 1-8, Method. for use in the preparation of hRSV infections prevention and / or pharmaceutical composition for the treatment, polypeptide according to any one of claims 1-8, or any of claims 11 to 13 one Use of a composition as described in paragraph. At least one of respiratory disease, upper respiratory tract infection, lower respiratory tract infection, bronchiolitis (inflammation of the peripheral airways in the lungs), pneumonia, dyspnea, cough, wheezing, asthma, recurrent wheezing and recurrent asthma for the prevention and / or treatment, a polypeptide according to any one of claims 1-8, or composition according to any one of claims 11 to 13. Polypeptide according to any one of claims 1-8, including 及 beauty / or compositions comprising the same, pharmaceutical devices for pulmonary delivery. 19. The pharmaceutical device according to claim 18 , which is an inhaler, aerosol or dry powder inhaler for suspensions of liquids, solid microparticles or droplets.